Chimeric, human and humanized anti-granulocyte antibodies and methods of use

ABSTRACT

The present invention provides humanized, chimeric and human MN3 antibodies, fusion proteins, and fragments thereof. The antibodies, fusion proteins, and fragments thereof, as well as combinations with other suitable antibodies, are useful for the treatment and diagnosis of granulocyte related disorders and diseases, such as leukemia.

CLAIM FOR PRIORITY

The present application is a non-provisional application of U.S.Provisional Patent Application Ser. No. 60/414,341, the entire contentsof which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to humanized, chimeric and humananti-granulocyte antibodies, particularly monoclonal antibodies (MAbs),therapeutic and diagnostic conjugates of humanized, chimeric and humananti-granulocyte antibodies and methods of diagnosing or treating amalignancy, inflammation, atherosclerosis, infarction or other diseasesmanifesting an increased presence of activated granulocytes, usinghumanized, chimeric and fully human anti-granulocyte antibodies.Preferred anti-granulocyte antibodies are those binding the NCA90 andNCA95 antigens, such as the MN3 monoclonal antibody against NCA90, theMabs MN-2, MN-15, NP-1, NP-2, BW 250/183 against NCA95, Mab 47, andantibodies directed to antigens present on a single granulocyteprecursor, such as anti-CD-15 and anti-CD-33, or a combination thereof.The present invention also relates to antibody fusion proteins orfragments thereof comprising at least two anti-granulocyte MAbs orfragments thereof or at least one anti-granulocyte MAb or fragmentthereof and at least one second MAb or fragment thereof, other than theanti-granulocyte MAb or fragment thereof. The humanized, chimeric andhuman anti-granulocyte MAbs, fragments thereof, antibody fusion proteinsthereof or fragments thereof may be administered alone, as a therapeuticconjugate or in combination with a therapeutic immunoconjugate, withother naked antibodies, or with therapeutic agents or as a diagnosticconjugate. The present invention further relates to DNA sequencesencoding humanized, chimeric and human MN3 antibodies against NCA 90,and antibody fusion proteins, vectors and host cells containing the DNAsequences, and methods of making the humanized, chimeric and human MN3antibodies.

The invention relates to immunological reagents for therapeutic use, forexample, in radioimmunotherapy (RAIT) and chemoimmunotherapy, anddetection and/or diagnostic uses, for example, in radioimmunodetection(RAID), ultrasonography, and magnetic resonance imaging (MRI). Inparticular, the invention relates to naked antibodies (unconjugated) anddirectly-conjugated antibodies, as well as bi-specific antibodies(bsAbs) and bi-specific antibody fragments (bsFabs) which have at leastone arm which is reactive against a targeted tissue and at least oneother arm which is reactive against a linker moiety. Further, theinvention relates to monoclonal antibodies that have been raised againstspecific immunogens, being human, humanized and chimeric monoclonalantibodies, as well as human, humanized and chimeric bi-specificantibodies and antibody fragments having at least one arm which isreactive against a targeted tissue or cell type and at least one otherarm which is reactive against a linker moiety, DNAs that encode suchantibodies and antibody fragments, and vectors for expressing the DNAs.

2. Background

Monoclonal antibodies (MAbs) have wide diagnostic and therapeuticpotentials in clinical practices against cancer and other diseases.Early clinical trials revealed encouraging results using radiolabeledMAbs for the diagnosis/detection (radioimmunodetection: RAID) andtreatment (radioimmunotherapy: RAIT) of malignancies in cancer patients(Goldenberg et al., (1993) (Intl. J. Oncol. 3:5-11; Goldenberg et al.,(1995) Immunol. Today 16:261-264; Goldenberg (1993) Am. J. Med.94:297-312; Goldenberg (1991) Adv. Exp. Med. Biol., 303:107-117).Monoclonal antibodies play a central role in cancer immunotherapy,either in naked forms, or as conjugates to cytotoxic agents, such asradioisotopes, drugs, toxins, or prodrug-converting enzymes (Goldenberget al., (1993) Immunol. Today, 14:5-7). These approaches are underactive evaluation, with different levels of developmental and clinicalsuccesses. Naked MAbs potentially may achieve clinical responses byinducing a cytotoxic effect upon binding to cell surface proteins thatare over-expressed on cancer cells. Studies have shown that thesetherapeutic effects were accomplished by controlling tumor growth viaprogrammed cell death (apoptosis), or by the induction of anti-tumorimmune responses (Cragg et al., (1999) Curr. Opin. Immunol.,11:541-547).

The majority of clinically interesting antibodies were raised in mice.The problem of immunogenicity of murine MAbs in humans has been themajor obstacle preventing their clinical application, especially incancer therapy where large doses and repeated administrations arerequired to achieve maximum efficacy. It has been demonstrated thatsignificant human-anti-mouse antibody (HAMA) responses were detected inapproximately 50% of patients after a single injection of murine MAb;greater than 90% of patients developed HAMA following two or threerepeated injections (Sears et al., (1984) J. Biol. Response Med.3:138-150; Reynolds et al., (1989) Int. J. Rad. Appl. Instrum. B,16:121-125; Shawler et al. (1985) J. Immunol., 135:1530-1535; Jaffers etal., (1986) Transplantation, 41:572-578). In addition, the therapeuticeffects of these murine MAbs in humans, if any, are further mitigatedwith their short serum half-lives and inabilities to recruit humaneffector cells, such as complement-fixing cytotoxic T cells. With theadvent of molecular engineering, we can now genetically modify thestructure of an antibody without affecting its antigen specificity tominimize or eliminate the HAMA responses and simultaneously enhance itsimmune effector functions. The processes are called chimerization andhumanization. These modified MAbs have been shown to possess attributesessential for enhanced clinical utility, i.e., decreasedimmunogenicities, longer serum half-lives in human, and the ability torecruit effector functions.

Granulocytes, including neutrophils, basophils and eosinophils, arewhite blood cells that help mediate the humoral immune response.Granulocytes play an important role in defense of the host organism bymigrating to sites of infection or injury and initiating phagocytosisand production of inflammatory mediators. One consequence of thisactivity is acute inflammation that can cause damage to the surroundingtissue. Abnormal granulocytes production, proliferation and/ordedifferentiation can also result in myeloid leukemia.

Inflammation has also been implicated as a major contributing factor incystic fibrosis. See Konstan, M. W. et al., Infection and Inflammationin the Lung in Cystic Fibrosis, in Cystic Fibrosis, Davis, P. B. (ed.),Marcel Dekker, Inc., NY (1993). The inflammatory response to thisinfection is excessive and persistent. It sets the stage for a viciouscycle of airway obstruction, infection, and inflammation that ultimatelyleads to lung destruction. See Davis, P. B. et al. Am. J. Respir. Crit.Care Med. 154:1229-1256 (1996) and Konstan, M. W. et al., Pediatr.Pulmonol. 24:137-142 (1997). The inflammatory component of CF ischaracterized by persistent infiltration of neutrophils, which includestimes of clinical stability. See Konstan, M. W. et al., Am. J. Respir.Crit. Care Med. 150:448-454 (1994). This occurs very early in the courseof the disease for many patients, frequently during the first year oflife, and may exist even in the absence of apparent infection. SeeKonstan, M. W. et al., Pediatr. Pulmonol. 24:137-142 (1997). Further, inacute myocardial infarction, particularly resulting from compromisedblood flow (ischemia because of vessel compromise), insipidation ofgranulocytes into the diseased myocardium results and plays a prominentrole in the tissue damage and infarction resulting from ischemia of themyocardium. In this situation, it has now been discovered that anantibody targeting such activated granulocytes not only can assist inthe diagnosis of extent of ischemic disease, but can in fact interruptthe progression of infarction and tissue necrosis by binding to thepathology-inducing, activated granulocytes.

Accordingly, there remains a need for imaging granulocyte populationsand their localization to determine sites of inflammation. There alsoremains a need for effective therapies of granulocyte disorders likemyeloid leukemias, as well as preventing progression of myocardialinfarction following myocardial ischemia.

SUMMARY OF THE INVENTION

The present invention provides a monoclonal antibody (MAb) or fragmentthereof that binds granulocyte (neutrophil) antigen. Preferably, thepresent invention provides humanized, chimeric and humananti-granulocyte-targeting antibodies, such as MN3 antibodies, which areuseful for the treatment and diagnosis of a malignancy and of diseasesresulting from the accumulation in tissue of activated granulocytes,such as in ischemic injury.

The present invention also provides a humanized MN3 (hMN3) monoclonalantibody (MAb) or fragment thereof comprising one or morecomplementarity-determining regions (CDRs) of a murine MN3 MAb and oneor more framework (FR) regions of the light and heavy chain variableregions of a human antibody and the light and heavy chain constantregions of a human antibody. The CDRs of the light chain variable regionof the humanized can be selected from a MN3 MAb CDR1 comprising aminoacids RSSQSIVHSNGNTYLE (SEQ ID NO: 1); a CDR2 comprising an amino acidsequence of KVSNRFS (SEQ ID NO: 2); and a CDR3 comprising an amino acidsequence of FQGSHVPPT (SEQ ID NO: 3). The CDRs of the heavy chainvariable region of the MN3 MAb can be selected from a CDR1 comprisingamino acids NYGMN (SEQ ID NO: 4); a CDR2 comprising amino acidsWINTYTGEPTYADDFKG (SEQ ID NO: 5); and a CDR3 comprising amino acidsKGWMDFNGSSLDY (SEQ ID NO: 6).

The invention further provides a humanized antibody molecule comprisinga variable domain wherein the complementarity determining regions (CDRs)of said variable domain are from the mouse monoclonal MN3 antibody andthe remainder of the immunoglobulin is from one or more humanimmunoglobulins.

Also provided by the present invention is a humanized antibody heavychain comprising a variable domain wherein the CDRs of said variabledomain are from the mouse monoclonal antibody MN3 heavy chain and theremainder of the immunoglobulin is from the heavy chain of one or morehuman immunoglobulins.

Also provided by the present invention is a CDR-grafted humanized heavychain comprising the complementarity determining regions (CDRs) of amurine MN3 MAb and the framework region of the heavy chain variableregion of a human antibody and the heavy chain constant region of ahuman antibody, wherein the CDRs of the heavy chain variable region ofthe humanized MN3 MAb comprises CDR1 comprising an amino acid sequenceof NYGMN (SEQ ID NO: 4); CDR2 comprising an amino acid sequence ofWINTYTGEPTYADDFKG (SEQ ID NO: 5) and CDR3 comprising an amino acidsequence of KGWMDFNGSSLDY (SEQ ID NO: 6).

A CDR-grafted humanized light chain comprising the complementaritydetermining regions (CDRs) of a murine MN3 MAb and the framework regionof the light chain variable region of a human antibody and the lightchain constant region of a human antibody, wherein the CDRs of the lightchain variable region of the humanized MN3 MAb comprises CDR1 comprisingan amino acid sequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 1); CDR2comprising an amino acid sequence of KVSNRFS (SEQ ID NO: 2)and CDR3comprising an amino acid sequence of FQGSHVPPT (SEQ ID NO: 3).

If the humanized antibody molecule is a CDR-grafted humanized antibodymolecule, appropriate variable region framework sequences may be usedhaving regard to class/type of the donor antibody from which the antigenbinding regions are derived. Preferably the type of human framework usedis of the same/similar class/type as the donor antibody. Advantageouslythe framework is chosen to maximise/optimize homology with the donorantibody sequence particularly at positions specially close or adjacentto the CDRs. Examples of human frameworks which may be used to constructCDR-grafted antibodies are LAY, POM, TUR, TEI, KOL, NEWM, REI and EU;for instance KOL and NEWM for the heavy chain and REI for the lightchain or EU for both the heavy chain and light chain.

In another embodiment, the humanized MN3 antibody or fragment thereofcomprises at least one amino acid substituted from the correspondingposition of the FR of the murine MN3 antibody or fragment thereof.Preferably, the murine amino acid from the murine MN3 MAb or fragmentthereof is at least one amino acid selected from the group consisting ofamino acid residue 27, 30, 67, 68, 69 or 94 of the murine heavy chainvariable region as numbered in FIG. 4B. Also preferred, the murine aminoacid from the murine MN3 MAb or fragment thereof is at least one aminoacid selected from the group consisting of amino acid residue 20, 22,39, 60, 70 or 100 of the murine light chain variable region shown inFIG. 4A.

In a preferred embodiment, the MN3 fragments of the present inventionare selected from the group consisting of Fv, F(ab′)₂, Fab′ and Fab, aswell as scFv and related single-chain, antigen-binding, constructs.

The invention also provides a humanized MN3 MAb or fragment thereofcomprising the hMN3Vk of FIG. 4B and/or the hMN3VH1 of FIG. 4A.

Further provided is a chimeric MN3 (cMN3) monoclonal antibody, orfragment thereof comprising the complementarity-determining regions(CDRs) of a murine MN3 MAb and the framework (FR) regions of the lightand heavy chain variable regions of said murine anti-CD 20 MAb and thelight and heavy chain constant regions of a human antibody, wherein theCDRs of the light chain variable region of the chimeric MN3 MAbcomprises CDR1 comprising an amino acid sequence RSSQSIVHSNGNTYLE (SEQID NO: 1); CDR2 comprising an amino acid sequence of KVSNRFS; and CDR3comprise an amino acid sequence of FQGSHVPPT (SEQ ID NO: 2); and theCDRs of the heavy chain variable region of the MN3 MAb comprise CDR1comprising amino acids NYGMN (SEQ ID NO: 4); CDR2 comprising amino acidsWINTYTGEPTYADDFKG (SEQ ID NO: 5) and CDR3 comprising amino acidsKGWMDFNGSSLDY (SEQ ID NO: 6).

The invention further provides a chimeric MN3 (cMN3) monoclonal antibody(MAb) or fragment thereof comprising the light and heavy chain variableregions of murine MN3 MAb and the light and heavy chain constant regionsof a human antibody, wherein said cMN3 comprises the light chainvariable region as set forth in FIG. 4B designated as cMN3Vk and theheavy chain variable region set forth in FIG. 4A designated as cMN3VH.

The invention also provides a human MN3 (MN3) monoclonal antibody (MAb)or fragment thereof comprising the light and heavy chain variable andconstant regions of a human antibody, wherein the CDRs of the lightchain variable region of the human MN3 MAb comprises CDR1 comprising anamino acid sequence RSSQSIVHSNGNTYLE (SEQ ID NO: 1); CDR2 comprising anamino acid sequence of KVSNRFS (SEQ ID NO: 2); and CDR3 comprise anamino acid sequence of FQGSHVPPT (SEQ ID NO: 3); and the CDRs of theheavy chain variable region of the MN3 MAb comprise CDR1 comprisingamino acids NYGMN (SEQ ID NO: 4); CDR2 comprising amino acidsWINTYTGEPTYADDFKG (SEQ ID NO: 5) and CDR3 comprising amino acidsKGWMDFNGSSLDY (SEQ ID NO: 6).

The invention further provides a method for the expression of an MN3 MAbor fragment thereof or antibody fusion protein or fragment thereof.

The invention also provides a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites havingaffinity toward an antigen recognized by MN3 and one or more bindingsites having affinity towards hapten molecules.

Also contemplated herein is a diagnostic/detection or therapeuticimmunoconjugate comprising an antibody component that comprises any ofthe MN3 MAbs or fragments thereof of the present invention, or anantibody fusion protein or fragment thereof that comprises any of theMN3 antibodies or fragments thereof of the present invention, whereinthe antibody component is bound to at least one diagnostic/detectionagent or at least one therapeutic agent. Similarly, any anti-granulocyteantibody performing similar targeting functions, such as an NCA95, aCD33, a CD15, or other such antibodies can be used as described for theanti-NCA90 MAb, MN3. Preferably, the diagnostic/detection or therapeuticagent of the immunoconjugate according to the present invention is boundto said MAb or fragment thereof by means of a carbohydrate moiety.

In some embodiments, the present compositions and methods are useful fordiagnosing or detecting granulocyte disorders, such as myeloid leukemiasand inflammation, including that caused by myocardial ischemia, cysticfibrosis, appendicitis, inflammatory bowel disease and pelvicinflammatory disease. The present methods can also be used to diagnoseor detect space-occupying lesions of the bone marrow, where a negativeuptake or image of the bone marrow using the present antibodies andfragments indicates the presence of the lesion. Detection of bone marrowlesions can be useful in determining if a metastatic cancer, such as aprostate, lung or breast cancer, has infiltrated the bone marrow. Thediagnosis or detection methods can be particularly useful for patientsknown or suspected of having such disorders, inflammation or malignancy.

In one embodiment, the diagnostic/detection immunoconjugate comprises atleast one photoactive diagnostic/detection agent, such as a chromagen ordye at least one radionuclide with an energy between 20 and 10,000 keV,such as a gamma-, beta- or a positron-emitting isotope, a contrastagent, such as a radiopaque compound, a paramagnetic ion, includingchromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III), or an ultrasound-enhancing agent,including a liposome that is conjugated to a humanized anti-granulocyteantibody or fragment thereof. The liposome can be gas-filled. Theradiopaque compound may be selected from the group consisting of iodinecompounds, barium compounds, gallium compounds and thallium compounds.In another embodiment, the diagnostic/detection described herein is usedin intraoperative, endoscopic, or intravascular detection/diagnosis.

Also contemplated herein is a therapeutic immunoconjugate comprising atherapeutic agent that is selected from the group consisting of aradionuclide, boron, gadolinium or uranium atoms, an immunomodulator,such as a cytokine, a stem cell growth factor, a lymphotoxin, such astumor necrosis factor (TNF), a hematopoietic factor such as aninterleukin (IL), a colony stimulating factor (CSF) such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), an interferon (IFN) suchas interferons-α, -β or -γ, and a stem cell growth factor such as thatdesignated “S1 factor,” a hematopoietic factor, erythropoietin,thrombopoietin, an antibody, a hormone, a hormone antagonist, an enzyme,an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxic drug,such as antimitotic, alkylating, antimetabolite,angiogenesis-inhibiting, apoptotic, alkaloid, COX-2-inhibiting andantibiotic agents, a cytotoxic toxin, such as plant, microbial, andanimal toxins, and a synthetic variations thereof, an angiogenesisinhibitor, a different antibody and a combination thereof. In apreferred embodiment, the cytokine is selected from the group consistingof IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferon-γ,TNF-α and a combination thereof, the radionuclide is selected from thegroup consisting of an Auger emitter, a beta-emitter and analpha-emitter, such as P-32, P-33, Sc-47, Fe-59, Cu-64, Cu-67, Se-75,As-77, Sr-89, Y-90, Mo-99, Rh-105, Pd-109, Ag-111, I-125, I-131, Pr-142,Pr-143, Pm-149, Sm-153, Tb-161, Ho-166, Er-169, Lu-177, Re-186, Re-188,Re-189, Ir-194, Au-198, Au-199, Pb-211, Pb-212, and Bi-213, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161,Os-189m, Ir-192, Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211,Ac-225, Fr-221, At-217, Bi-213, Fm-255, B-10, Gd-157, U-235, andcombinations thereof. Preferably, the radionuclide has an energy between20 and 10,000 keV.

In another embodiment, the therapeutic agent conjugated to theanti-granulocyte antibody or fragment thereof is a photoactivetherapeutic agent, such as a chromogen or dye.

Considered in the present invention also is a multivalent, multispecificantibody or fragment thereof comprising one or more antigen-bindingsites having affinity toward an antigen recognized by theanti-granulocyte antibody and one or more hapten binding sites havingaffinity towards hapten. Preferably, the anti-granulocyte antibodyantibody or fragment thereof is humanized. Also preferred, the antibodyor fragment thereof is fully human or chimerized. In one embodiment, themultivalent, multispecific antibody or fragment thereof comprises adiagnostic/detection or therapeutic agent.

Also considered in the present invention is an antibody fusion proteinor fragment thereof comprising at least two anti-granulocyte MAbs orfragments thereof, wherein the MAbs or fragments thereof are selectedfrom any of the NCA90, NCA95, CD33, or CD15 monoclonal antibodies orfragments thereof of the present invention. In a similar vein, anantibody fusion protein or fragment thereof comprising at least onefirst anti-granulocyte MAb or fragment thereof of any one theanti-granulocyte antibodies or fragments of the present invention, andat least one second MAb or fragment thereof, other than any one of thefirst anti-granulocyte MAbs or fragments thereof of the presentinvention, is also contemplated. In a preferred embodiment, the secondMAb is another, but different granulocyte-associated antibody. Inanother preferred embodiment, the antibody fusion protein or fragmentthereof further comprises a diagnostic/detection or therapeutic agentconjugated to the fusion protein or fragment thereof.

Considered herein is a method of treating a malignancy or other diseaseinvolving accumulation of activated or neoplastic granulocytes in asubject, comprising the step of administering to said subject atherapeutically effective amount of a naked and/or conjugatedanti-granulocyte antibody, fusion protein, or fragment thereof of thepresent invention, formulated in a pharmaceutically acceptable vehicle,either alone or in combination with other therapeutic and/or diagnosticagents. Preferably, a method of treating a malignancy in a subject,comprising the step of administering to said subject a therapeuticallyeffective amount of an immunoconjugate or fragment thereof the presentinvention, formulated in a pharmaceutically acceptable vehicle.

Similarly, a method of diagnosing/detecting a malignancy or othergranulocyte-related disease in a subject, comprising the step ofadministering to said subject a diagnostically effective amount of anaked or conjugated anti-granulocyte antibody, fusion protein, orfragment thereof of the present invention, optionally formulated in apharmaceutically acceptable vehicle. These methods can further involvethe step of detecting whether the anti-granulocyte antibody binds to thetarget antigen.

Yet another embodiment of the present invention provides a method forthe ablation of bone marrow comprising administering to a subject one ormore of the antibodies or fragments described herein coupled to a bonemarrow ablation agent.

Another embodiment is a method of treating or diagnosing/detecting amalignancy in a subject, comprising (i) administering to a subject inneed thereof the anti-granulocyte antibody or fragments thereof of thepresent invention; (ii) waiting a sufficient amount of time for adesired amount of the non-binding protein to clear the subject'sbloodstream; and (iii) administering to said subject a carrier moleculecomprising a diagnostic agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of said antibody.

The present compositions also include nucleic acids encoding thedisclosed antibodies to NCA90 (MN3), such as those shown in the figures,vectors containing these nucleic acids and cells containing the nucleicacids. The present invention also provides methods for producing the MN3Mabs disclosed herein using the nucleic acids, vectors and transfectedcells.

Another embodiment of the present invention is a DNA sequence and avector comprising a DNA sequence, and a host cell comprising a DNAsequence, that comprises a nucleic acid encoding an MN3 MAb or fragmentthereof selected from the group consisting (a) an MN3 MAb or fragmentthereof of the present invention; (b) an antibody fusion protein orfragment thereof comprising at least two of said MAbs or fragmentsthereof; (c) an antibody fusion protein or fragment thereof comprisingat least one first MN3 MAb or fragment thereof comprising said MAb orfragment thereof of any one of the antibodies of the present inventionand at least one second MAb or fragment thereof, other than the MN3 MAbor fragment thereof described in the present invention; and (d) anantibody fusion protein or fragment thereof comprising at least onefirst MAb or fragment thereof comprising said MAb or fragment thereof ofany one of the antibodies of the present invention and at least onesecond MAb or fragment thereof, other than the MN3 MAb or fragmentthereof of any one of the antibodies of the present invention, whereinsaid second MAb is selected from the group consisting of anti-NCA-90,anti-NCA-95, MN-2, MN-3, MN-15, NP-1, NP-2, BW 250/183, and MAb 47 andantibodies directed to antigens present on a single granulocyteprecursor, such as anti-CD-15 and anti-CD-33, or a combination thereof.

A method of delivering a diagnostic/detection or therapeutic agent, or acombination thereof, to a target comprising (i) providing a compositioncomprising an immunoconjugate that comprises the antibody, fusionprotein, or fragment thereof of any one of the antibodies, fusionproteins, or fragments thereof of the present invention and (ii)administering to a subject in need thereof said composition, is alsodescribed. Preferably, the diagnostic/detection agent comprises at leastone photoactive diagnostic agent, such as a chromogen or dye, a contrastagent, such as a paramagnetic ion, an ultrasound-enhancing agent or aradiopaque compound used in X-rays or computed tomography, such as aniodine compound, barium compound, gallium compound or thallium compound.In one embodiment, the ultrasound-enhancing agent is a liposome thatcomprises a humanized anti-granulocyte antibody or fragment thereof, andoptionally, the liposome is gas-filled. In another embodiment, thediagnostic/detection agent preferably is a radionuclide with an energybetween 20 and 2,000 keV, such as a gamma-, beta- or a positron-emittingisotope. Still preferred, the radionuclide is selected from the groupconsisting of F-18, Mn-51, Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68,As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120,I-124, Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m,In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197, and Tl-201. Alsopreferred, the radiopaque compound is selected from the group consistingof barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

Similarly, in the method of delivering a diagnostic/detection ortherapeutic agent, or a combination thereof, to a target, thetherapeutic agent is preferably selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an enzyme inhibitor, a photoactive therapeutic agent, acytotoxic agent, such as a drug or toxin (including a plant, microbialand animal toxin, and a synthetic variation thereof), and a combinationthereof. Preferably, the drug is selected from the group consisting ofantimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,anthracyclines, alkaloid, COX-2-inhibitor and antibiotic agents, andcombinations thereof, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, pyrimidine analogs, purine analogs, antibiotics, enzymes,enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes,vinca alkaloids, substituted ureas, methyl hydrazine derivatives,adrenocortical suppressants, hormones, hormone antagonists, endostatin,taxols, camptothecins, doxorubicins and their analogs, and a combinationthereof. Also preferred, the toxin is selected from the group consistingof ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.Suitable enzymes include malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

Also considered herein is a method of delivering a diagnostic/detectionagent, a therapeutic agent, or a combination thereof to a target,comprising: (i) administering to a subject a multivalent, multispecificantibody or fragment thereof of the present invention; (ii) waiting asufficient amount of time for an amount of the non-binding protein toclear the subject's blood stream; and (iii) administering to saidsubject a carrier molecule comprising a diagnostic/detection agent, atherapeutic agent, or a combination thereof, that binds to a bindingsite of said antibody. Preferably, the multivalent, multispecificantibody or fragment thereof comprises one or more antigen-binding siteshaving affinity toward an antigen recognized by MN3 and one or morehapten binding sites having an affinity towards hapten molecules.Preferably, the carrier molecule binds to more than one binding site ofthe antibody. Also preferred, the diagnostic/detection agent or saidtherapeutic agent is selected from the group comprising isotopes, dyes,chromogens, contrast agents, drugs, toxins, cytokines, enzymes, enzymeinhibitors, hormones, hormone antagonists, growth factors,radionuclides, and metals.

Contemplated herein is a method of treating a malignancy or anothergranulocyte-associated disease in a subject comprising administering tosaid subject a therapeutically effective amount of (i) an antibody orfragment thereof or (ii) an antibody fusion protein or fragment thereof,wherein the antibody or fragment thereof comprises at least two MAbs orfragments thereof, at least one of which is any of the anti-granulocyteMAbs or fragments thereof of the present invention, and the fusionprotein or fragment thereof comprises at least one an binding site foran antigen recognized by the anti-granulocyte MAb, formulated in apharmaceutically suitable excipient. In a preferred embodiment, at leastone of the Mabs or fragments thereof is a naked Mab or fragment thereof.In another embodiment, the fusion protein comprises a second bindingsite that is reactive with a tumor marker substance or a granulocytetarget antigen other than an antigen recognized by the first antibody.Also contemplated is that the anti-granulocyte antibody or fragmentthereof, or anti-granulocyte MAb fusion protein or fragment thereof, isadministered before, concurrently, or after at least one therapeutic ordiagnostic/detection agent.

Another embodiment is a method of treating a malignancy or agranulocyte-related disease in a subject comprising administering tosaid subject a therapeutically effective amount of an antibody orfragment thereof comprising at least two MAbs or fragments thereof,wherein the MAbs are selected from any one of the anti-granulocyteantibodies described herein, and formulated in a pharmaceuticallysuitable excipient. In a preferred embodiment, at least one of the Mabsor fragments thereof is a naked Mab or fragment thereof. Alsocontemplated is that the anti-granulocyte antibody or fragment thereof,or the anti-granulocyte fusion protein or fragment thereof, isadministered before, concurrently, or after at least one therapeuticand/or diagnostic/detection agent.

In the method of treatment described herein, the MN3 antibody isselected from a chimeric MN3 antibody, human MN3 antibody, and humanizedMN3 antibody. Preferably, the chimeric, human and humanized MN3 antibodyconstant and hinge regions comprise constant and hinge regions of ahuman IgG1. Also in the methods described herein, the MN3 antibody orfragment thereof or fusion protein or fragment thereof is administeredbefore, in conjunction with, or after a second conjugated antibodyreactive with a second tumor marker or an activated granulocyte antigentarget expressed by said malignancy or granulocyte-associated disease,respectively, is administered to said subject.

The present invention also describes a method of diagnosing or detectinga malignancy or an ischemic lesion in a subject comprising administeringto said subject a diagnostically effective amount of adiagnostic/detecting conjugate comprising a MN3 MAb or fragment thereofor a fusion protein or fragment thereof of as described in the presentinvention, wherein the MN3 MAb or fragment thereof, or fusion protein orfragment thereof, is bound to at least one diagnostic/detection agent,formulated in a pharmaceutically suitable excipient.

Another embodiment of the present invention is a method of treating amalignant myeloid cell population or an ischemic injury in a subjectcomprising (i) administering to said subject a therapeutically effectiveamount of a composition comprising a naked or conjugatedanti-granulocyte MAb or fragment thereof or a naked or conjugatedantibody fusion protein or fragment thereof, as described in the presentinvention, and (ii) optionally formulating said anti-granulocyte MAb orfragment thereof or antibody fusion protein or fragment thereof in apharmaceutically suitable excipient. Preferably, the anti-granulocyteantibody, fusion protein, or fragment thereof is an MN3 antibody, fusionprotein, or fragment thereof. Optionally, the composition may furthercomprise a second naked or conjugated antibody or fragment thereof, ornaked or conjugated antibody fusion protein or fragment thereof, thatmay or be an MN3 antibody, fusion protein or fragment thereof, or maybind a second marker expressed by the malignancy or ischemic lesion.Also considered is that the anti-granulocyte antibody, antibody fusionprotein, or fragment thereof, is administered before, in conjunctionwith, or after a second antibody, fusion protein, or fragment thereof isadministered to said subject. The anti-granulocyte antibody may also beadministered before, concurrently or after a therapeutic ordiagnostic/detection agent.

The present invention also describes a method of diagnosing or detectinga malignancy in a subject comprising (i) performing an in vitrodiagnosis assay on a specimen from the subject with a compositioncomprising an anti-granulocyte MAb or fragment thereof or an antibodyfusion protein or fragment thereof described herein. Preferably themalignancy is a granulocyte, e.g. a neutrophil, expressing an antigenrecognized, for example, by MN3, such as a myeloid leukemia. Alsopreferred, the in vitro diagnosis assay is selected from the groupconsisting of immunoassays, RT-PCR and immunohistochemistry. If thediagnostic assay is RT-PCR or immunoassays, the specimen is preferablybody fluid or a tissue or cell population. If the diagnostic assay isimmunohistochemistry or immunocytochemistry, the specimen is preferablya cell aliquot or a tissue.

In any of the methods of the present invention, the subject ispreferably a mammal, such as a human or domestic pet.

Another embodiment of the present invention is a method of treating oridentifying diseased tissues in a subject, comprising: (A) administeringto said subject a bi-specific antibody or antibody fragment having atleast one arm that specifically binds a diseased tissue-associatedmarker and at least one other arm that specifically binds a targetableconjugate, wherein said diseased tissue-associated marker is an antigenrecognized by the anti-granulocyte MAb; (B) optionally, administering tosaid subject a clearing composition, and allowing said composition toclear non-localized antibodies or antibody fragments from circulation;(C) administering to said subject a first targetable conjugate whichcomprises a carrier portion which comprises or bears at least oneepitope recognizable by said at least one other arm of said bi-specificantibody or antibody fragment, and one or more conjugated therapeutic ordiagnostic agents; and (D) when said therapeutic agent is an enzyme,further administering to said subject (i) a prodrug, when said enzyme iscapable of converting said prodrug to a drug at the target site; or (ii)a drug which is capable of being detoxified in said subject to form anintermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or (iii) a prodrug which is activated in said subject through naturalprocesses and is subject to detoxification by conversion to anintermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or (iv) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by said atleast one other arm of said bi-specific antibody or antibody fragment,and a prodrug, when said enzyme is capable of converting said prodrug toa drug at the target site. Preferably, at least one arm thatspecifically binds a targeted tissue is a human, chimeric or humanizedanti-granulocyte antibody or a fragment of a human, chimeric orhumanized anti-granulocyte antibody. Also preferred, the targetableconjugate comprises at least two HSG (histamine-succinyl-glycine)haptens. Preferably, the targeted tissue is a tumor or ischemic lesionwith an accumulation of granulocytes, and more preferably, the tumor orischemia produces or is associated with an antigen recognized by theanti-granulocyte antibody. Also preferred, the anti-granulocyte antibodyor fragment thereof comprises the Fv of the MAb. A preferred embodimentis the use of the MN3 MAb in such applications as a chimeric, humanized,or human antibody, as described herein.

This method may further comprise, when said first targetable conjugatecomprises a prodrug, administering a second targetable conjugate whichcomprises a carrier portion which comprises or bears at least oneepitope recognizable by said at least one other arm of said bi-specificantibody or antibody fragment, and an enzyme capable of converting saidprodrug to a drug or of reconverting a detoxified intermediate of saiddrug to a toxic form. Preferably, the prodrug is selected from the groupconsisting of epirubicin glucuronide, CPT-11, etoposide glucuronide,daunomicin glucuronide and doxorubicin glucuronide. Also preferred, thetargetable conjugate comprises one or more radioactive isotopes usefulfor killing diseased tissue. The targetable conjugate may comprise oneor more agents for photodynamic therapy, such as a photosensitizer. In apreferred embodiment, the photosensitizer is selected from the groupconsisting of benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin(SnET2), sulfonated aluminum phthalocyanine (AlSPc) and lutetiumtexaphyrin (Lutex).

Considered herein is a method for detecting or treating tumors orischemic lesions expressing an antigen recognized by an anti-granulocyteMAb in a mammal, comprising: (A) administering an effective amount of abispecific antibody or antibody fragment comprising at least one armthat specifically binds a targeted tissue and at least one other armthat specifically binds a targetable conjugate, wherein said one armthat specifically binds a targeted tissue is an anti-granulocyteantibody or fragment thereof; and (B) administering a targetableconjugate. The targetable conjugate can be selected from the groupconsisting of (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂(SEQ ID NO: 7); (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂; (iv)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂; (v)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vi)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vii)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (viii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; (ix)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH₂; (x)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH₂; (xi)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH₂; (xii)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiv)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH₂; (xv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvi)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvii)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH₂; (xviii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH₂; (xix)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(TscG-Cys)-NH₂; (xx)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(TscG-Cys)-NH₂;

Additional targetable conjugates that can be used with the presentmethods include those disclosed in U.S. Patent Application No.60/478,403.

Preferably, the method further comprises administering to the subject aclearing composition, and allowing said composition to increaseclearance of non-localized antibodies or antibody fragments fromcirculation.

Also contemplated herein is a kit useful for treating or identifyingdiseased tissues involving accumulation of normal or malignantgranulocytes in a subject comprising: (A) a bi-specific antibody orantibody fragment having at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds atargetable conjugate, wherein said one arm that specifically binds atargeted tissue is an anti-granulocyte antibody or fragment thereof; (B)a first targetable conjugate which comprises a carrier portion whichcomprises or bears at least one epitope recognizable by said at leastone other arm of said bi-specific antibody or antibody fragment, and oneor more conjugated therapeutic or diagnostic agents; and (C) optionally,a clearing composition useful for clearing non-localized antibodies andantibody fragments; and (D) optionally, when said therapeutic agentconjugated to said first targetable conjugate is an enzyme, (i) aprodrug, when said enzyme is capable of converting said prodrug to adrug at the target site; or (ii) a drug which is capable of beingdetoxified in said subject to form an intermediate of lower toxicity,when said enzyme is capable of reconverting said detoxified intermediateto a toxic form, and, therefore, of increasing the toxicity of said drugat the target site, or (iii) a prodrug which is activated in saidsubject through natural processes and is subject to detoxification byconversion to an intermediate of lower toxicity, when said enzyme iscapable of reconverting said detoxified intermediate to a toxic form,and, therefore, of increasing the toxicity of said drug at the targetsite, or (iv) a second targetable conjugate which comprises a carrierportion which comprises or bears at least one epitope recognizable bysaid at least one other arm of said bi-specific antibody or antibodyfragment, and a prodrug, when said enzyme is capable of converting saidprodrug to a drug at the target site. Preferably, the targetableconjugate is selected from the group consisting of:

(i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂; (iv)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂; (v)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vi)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vii)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (viii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; (ix)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH₂; (x)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH₂; (xi)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH₂; (xii)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiv)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG) -D-Lys(DOTA)-NH₂; (xv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvi)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvii)Ac-D-Cys-D-Lys(DOTA)-D -Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH₂; (xviii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA) -NH₂; (xix)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(TscG-Cys)-NH₂; (xx) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(TscG-Cys)-NH₂;

Also described in the present invention is a method of screening for atargetable conjugate comprising: (A) contacting said targetableconstruct with a bi-specific antibody or antibody fragment having atleast one arm that specifically binds a marker associated with atargeted tissue, wherein said marker is an antigen recognized by MN3,and at least one other arm that specifically binds said targetableconjugate to give a mixture; and (B) optionally incubating the mixture;and (C) analyzing the mixture.

Another embodiment is a method for imaging malignant or ischemic tissueor cells in a mammal expressing an antigen recognized by ananti-granulocyte MAb, comprising: (A) administering an effective amountof a bispecific antibody or antibody fragment comprising at least onearm that specifically binds a marker associated with a targeted tissueand at least one other arm that specifically binds a targetableconjugate, wherein said marker is an antigen recognized by theanti-granulocyte MAb; and (B) administering a targetable conjugateselected from the group consisting of

(i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG) -NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂; (iv) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂; (v) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vi) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vii)DOTA-D-Ala-D -Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (viii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; (ix)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH₂; (x)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D -Lys(DTPA)-NH₂; (xi)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH₂; (xii) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr -D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiv)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG) -D-Lys(DOTA)-NH₂; (xv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvi)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvii)Ac-D-Cys-D-Lys(DOTA)-D -Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH₂; (xviii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA) -NH₂; (xix)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(TscG-Cys)-NH₂; (xx) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(TscG-Cys)-NH₂;

The invention also contemplates a method of intraoperativelyidentifying/disclosing diseased tissues expressing an antigen recognizedby an anti-granulocyte MAb, in a subject, comprising: (A) administeringan effective amount of a bispecific antibody or antibody fragmentcomprising at least one arm that specifically binds an antigenrecognized by the anti-granulocyte MAb and at least one other arm thatspecifically binds a targetable conjugate, wherein said one arm thatspecifically binds a targeted tissue is an anti-granulocyte MAb antibodyor fragment thereof; and (B) administering a targetable conjugateselected from the group consisting of

(i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG) -NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂; (iv) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂; (v) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vi) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vii)DOTA-D-Ala-D -Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (viii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; (ix)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH₂; (x)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D -Lys(DTPA)-NH₂; (xi)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH₂; (xii) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr -D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiv)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG) -D-Lys(DOTA)-NH₂; (xv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvi)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvii)Ac-D-Cys-D-Lys(DOTA)-D -Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH₂; (xviii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA) -NH₂; (xix)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(TscG-Cys)-NH₂; (xx) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(TscG-Cys)-NH₂;

Also described herein is a method for the endoscopic identification ofdiseased tissues expressing an antigen recognized by an anti-granulocyteMAb, in a subject, comprising: (A) administering an effective amount ofa bispecific antibody or antibody fragment comprising at least one armthat specifically binds an antigen recognized by an anti-granulocyte MAband at least one other arm that specifically binds a targetableconjugate wherein said one arm that specifically binds a targeted tissueis a MN3 antibody or fragment thereof; and (B) administering atargetable conjugate selected from the group consisting of

(i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG) -NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂; (iv) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂; (v)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vi)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vii) DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (viii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; (ix)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH₂; (x)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D -Lys(DTPA)-NH₂; (xi)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH₂; (xii) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr -D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiv)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG) -D-Lys(DOTA)-NH₂; (xv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvi)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvii)Ac-D-Cys-D-Lys(DOTA)-D -Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH₂; (xviii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA) -NH₂; (xix)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(TscG-Cys)-NH₂; (xx) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(TscG-Cys)-NH₂;

Another embodiment is a method for the intravascular identification ofdiseased tissues expressing an antigen recognized by an anti-granulocyteMAb, in a subject, comprising: (A) administering an effective amount ofa bispecific antibody or antibody fragment comprising at least one armthat specifically binds an antigen recognized by the anti-granulocyteMAb and at least one other arm that specifically binds a targetableconjugate wherein said one arm that specifically binds a targeted tissueis a MN3 antibody or fragment thereof; and (B) administering atargetable conjugate selected from the group consisting of

(i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG) -NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂; (iv) DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂; (v) DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vi) DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (vii)DOTA-D-Ala-D -Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (viii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; (ix)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH₂; (x)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D -Lys(DTPA)-NH₂; (xi)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH₂; (xii) Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr -D-Lys(HSG)-D-Lys(Tscg-Cys)-NH₂; (xiv)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG) -D-Lys(DOTA)-NH₂; (xv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvi)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂; (xvii)Ac-D-Cys-D-Lys(DOTA)-D -Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH₂; (xviii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA) -NH₂; (xix)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(TscG-Cys)-NH₂; (xx) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(TscG-Cys)-NH₂;

Another embodiment is a method of detecting lesions, preferably duringan endoscopic, laparoscopic, intravascular catheter, or surgicalprocedure, wherein the method comprises: (A) injecting a subject who isto undergo such a procedure with a bispecific antibody F(ab)₂ or F(ab′)₂fragment, or single-chain Fv fragment, wherein the bispecific antibodyor fragment has a first antibody binding site which specifically bindsto an antigen recognized by an anti-granulocyte MAb antigen, and has asecond antibody binding site which specifically binds to a hapten, andpermitting the antibody fragment to accrete at target sites; (B)optionally clearing non-targeted antibody fragments using agalactosylated anti-idiotype clearing agent if the bispecific fragmentis not largely cleared from circulation within about 24 hours ofinjection, and injecting a bivalent labeled hapten, which quicklylocalizes at the target site and clears through the kidneys; (C)detecting the presence of the hapten by close-range detection ofelevated levels of accreted label at the target sites with detectionmeans, within 48 hours of the first injection, and conducting saidprocedure, wherein said detection is performed without the use of acontrast agent or subtraction agent. In a preferred embodiment, thehapten is labeled with a diagnostic/detection radioisotope, a MRIimage-enhancing agent, a fluorescent label or a chemiluminescent label.Fluorescent labels can include rhodamine, fluorescein, renographin,fluorescein isothiocyanate, phycoerytherin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine. Chemiluminescentlabels can include luminol, isoluminol, an aromatic acridinium ester, animidazole, an acridinium salt and an oxalate ester.

Also considered is a method for close-range lesion detection, preferablyduring an operative, intravascular, laparoscopic, or endoscopicprocedure, wherein the method comprises: (A) injecting a subject to sucha procedure parenterally with an effective amount of an anti-granulocyteMAb immunoconjugate or fragment thereof, (B) conducting the procedurewithin 48 hours of the injection; (C) scanning the accessed interior ofthe subject at close range with a detection means for detecting thepresence of said labeled antibody or fragment thereof, and (D) locatingthe sites of accretion of said labeled antibody or fragment thereof bydetecting elevated levels of said labeled antibody or fragment thereofat such sites with the detection means. Preferably, the anti-granulocyteMAb immunoconjugate or fragment thereof comprises a radioisotope thatemits at an energy of 20-1,000 keV. Also preferred, the radioisotope isselected from the group consisting of technetium-99m, iodine-125,iodine-131, iodine-123, indium-111, fluorine-18, gallium-68 andgallium-67. In another embodiment, the anti-granulocyte MAbimmunoconjugate or fragment thereof comprises a non-isotopic agent, suchas a photoactive agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cloned V_(H) and Vκ gene sequences of the murine MN3 byRT-PCR and the deduced amino acid sequences. Underlined arrows at 5′-and 3′-ends indicate the sequence of PCR primers used in cloning. FIG.1A shows the DNA (SEQ ID NO: 8) and amino acid (SEQ ID NO: 9) sequencesof the MN3Vκ. FIG. 1B shows the DNA (SEQ ID NO: 10) and amino acid (SEQID NO: 11) sequences of the MN3V_(H). Amino acid sequences encoded bythe corresponding DNA sequences are given as one letter codes below thenucleotide sequence. Numbering of the nucleotide sequence is on theright side. The amino acid residues in the putative CDR regions areshown in bold and underlined. Kabat's Ig molecule numbering is used foramino acid residues as shown by the numbering above the amino acidresidues. The residues numbered by a letter following digits indicatethe insertion residues defined by Kabat numbering scheme. The insertionresidues numbered with a letter only have the same preceding digits asthe previous one. For example, residues 27A, 27B, 27C, 27D, and 27E inFIG. 1A are indicated as 82, A, B, and C27A, B, C, D, and E,respectively.

FIG. 2 shows the DNA and amino acid sequences of the chimeric MN3 (cMN3)heavy and light chain variable regions. FIG. 2A shows the DNA (SEQ IDNO: 12) and amino acid (SEQ ID NO: 13) sequences of the cMN3Vκ. FIG. 2Bshows the DNA (SEQ ID NO: 14) and amino acid (SEQ ID NO: 15) sequencesof the cMN3VH. Amino acid sequences encoded by the corresponding DNAsequences are given as one letter codes. The amino acid residues in theCDR regions are shown in bold and underlined. Numbering of thenucleotide sequence is on the right side. The numbering of amino acidsis same as that in FIG. 1.

FIG. 3 shows the results of a competitive ELISA assay to compare thebinding specificity and affinity of a chimeric MN3 (cMN3) with that ofmurine MN3 (mMN3). Varying concentrations of cMN3 (squares and solidline) or mMN3 (circles and dashed line) were mixed with a constantamount of biotinylated murine MN3 and incubated in microplate wellscoated with CEA. The residual binding of the biotinylated MN3 wasmeasured by HRP-conjugated streptavidin and substrate. The resultsshowed that cMN3 and the murine MN3 were comparable in their bindingtarget antigen.

FIG. 4 shows the alignment of the amino acid sequences of light andheavy chain variable regions of certain human antibodies, MN3 and hMN3.FIG. 4A compares the amino acid sequences of the REI (SEQ ID NO: 16),MN3 (SEQ ID NO: 17) and hMN3 (SEQ ID NO: 18) light chain variabledomains. FIG. 4B is the amino acid sequence alignment of EU (FR1-3) (SEQID NO: 19) and KOL (FR4) (SEQ ID NO: 22), MN3 (SEQ ID NO: 20) and hMN3(SEQ ID NO: 21) heavy chain variable domains. Boxed regions representthe CDR regions. Dots indicate the residues in MN3 and hMN3 which areidentical to the corresponding residues in REI Vκ. Dashes represent gapsintroduced to aid the alignment. Both N- and C-terminal residues(underlined) of hMN3 are fixed by the staging vector used. Thecorresponding terminal residues of MN3 are not compared with that of thehuman sequences. Kabat's Ig molecule numbering scheme is used (same asin FIGS. 1A and 1B, respectively).

FIG. 5 discloses the nucleotide sequences of hMN3Vκ (SEQ ID NO: 23) andthe adjacent flanking regions of the light chain staging vector, VKpBR2(SEQ ID NO: 24) (FIG. 5A) and hMN3VH (SEQ ID NO: 25) and the adjacentflanking regions of the heavy chain staging vector, VKpBS2 (SEQ ID NO:26) (FIG. 5B). The encoded amino acid sequences are shown as one lettercodes below the corresponding DNA sequences. The non-translatednucleotide sequences are shown in lowercase. The restriction sites usedfor subcloning are underlined and indicated. The secretion signalpeptide sequence is indicated by a double underline. Numbering of Vk andVH amino acid residues is the same as that in FIG. 2.

FIG. 6 shows the results of a competitive ELISA assay to compare thebinding activity of MN3, cMN3 and hMN3. Varying concentrations of cMN3(closed circles), hMN3 (closed squares) or mMN3 (closed triangles) weremixed with a constant amount of biotinylated murine MN3 and incubated inmicroplate wells coated with CEA. The residual binding of thebiotinylated MN3 was measured by HRP-conjugated streptavidin andsubstrate. The results indicated that cMN3 and hMN3 and the murine MN3antibody, MN3 competed equally well for the binding of antigen.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The present invention provides murine, humanized, chimeric and humananti-granulocyte, e.g., anti-neutrophil (granulocyte) antibodies, fusionproteins, or fragments thereof useful for treatment and/or diagnosis ofmammalian subjects, as an immunoconjugate or in combination with, butunconjugated to, other therapeutic and/or diagnostic agents. In apreferred embodiment, the anti-granulocyte antibody is an MN3 antibody.The MN3 antibodies and fragments thereof bind an antigen ongranulocytes. In some embodiments, the antibody or fragment thereof isnot a chimeric mouse-human antigranulocyte monoclonal antibody, such asis described by Sarwar et al., Radiat. Med. 16(5):391-7 (1998) or anantibody described by Zhao et al., J. Immunol. Methods, 270 (1):27-35(2002).

A preferred example of such an antibody is MN-3. See Hansen et al.,Cancer 71:3478-3485 (1993); Becker et al., Semin. Nucl. Med.24(2):142-53 (1994).

The MN-3 antibody was isolated from hybridomas derived from BALB/c micewhich were immunized with partially purified carcinoembryonic antigen(CEA) derived from GW-39 human colon adenocarcinoma xenografts. SeeHansen et al., Cancer 71:3478-3485 (1993). The MN-3 antibody is specificfor the NCA-90 antigen, a homotypic adhesion molecule expressed ongranulocytes, as well as normal colonic mucosa and colonicadenocarcinoma. See Becker et al., Semin. Nucl. Med. 24(2):142-53(1994); Watt et al., Blood 78:63-74 (1991).

Suitable amounts of the NCA-90 antigen, also referred to as CD66c, canbe obtained using standard techniques well-known in the art. Forexample, NCA-90 protein can be obtained from transfected cultured cellsthat overproduce NCA-90. Expression vectors that comprise DNA moleculesencoding NCA-90 can be constructed using the published NCA-90 nucleotidesequence. See Oikawa et al., Biochem. Biophys. Res. Commun. 146:464-460(1987); Wilson et al., J. Exp. Med. 173:137 (1991); Wilson et al., J.Immunol. 150:5013 (1993).

A variety of anti-granulocyte antibodies directed to antigens associatedwith various cell-types of the granulocyte/neutrophil can be used in thepresent invention. In one embodiment, the inventive methods utilizeanti-NCA-90 antibodies. In another embodiment, anti-NCA-95 antibodies,anti-CD-33, or anti-CD-15 antibodies are used. See Thakur et al., J.Nucl. Med., 37:1789-95 (1996); Ball et al., J. Immunol., 30:2937-41(1983); PCT WO 02/12347, incorporated in their entirety herein byreference. In still other embodiments, MN-2 and NP-2, which are classIIA anti-CEA antibodies, and MN-15 and NP-1, which are class I anti-CEAantibodies, are utilized. See Hansen et al., Cancer 71:3478-3485 (1993);Primus et al., Cancer Res. 43:686-692 (1983). Furthermore, BW 250/183 (amurine anti-NCA-95 antibody), and MAb 47 can be utilized. See Bosslet etal., Int. J. Cancer, 36:75-84 (1985); Meller et al., J. Nucl. Med.39:1248-1253 and Audette et al., Mol. Immunol. 24:1177-1186 (1987).Human and chimeric forms of these antibodies are preferred, andfull-human and humanized versions are most preferred. Subhuman primateantibodies and murine monoclonal antibodies may also be utilized.Constructs of multispecific and/or multivalent scFv constructs are alsosuitable for this invention.

Another suitable antibody is the MN-2 monoclonal antibody. The MN-2antibody was isolated from hybridomas derived from BALB/c mice whichwere immunized with partially purified carcinoembryonic antigen (CEA)derived from GW-39 human colon adenocarcinoma xenografts. See Hansen etal., Cancer 71:3478-3485 (1993). As a class IIA anti-CEA antibody, MN-2can be identified readily using blocking assays well-known in the art.See U.S. Pat. No. 4,818,709, which is hereby incorporated by referencein its entirety.

Another suitable antibody is the MN-15 monoclonal antibody. The MN-15antibody displays cross-reactivity between NCA-90 and NCA-95. MN-15 wasisolated from hybridomas derived from BALB/c mice which were immunizedwith partially purified carcinoembryonic antigen (CEA) derived fromGW-39 human colon adenocarcinoma xenografts. See Hansen et al., Cancer71:3478-3485 (1993). As a class I anti-CEA antibody, MN-15 can beidentified readily using blocking assays well-known in the art.

Still another suitable antibody is the NP-2 monoclonal antibody. TheNP-2 has specificity similar to that of MN-2. NP-2 was isolated fromhybridomas derived from BALB/c mice which were immunized with partiallypurified carcinoembryonic antigen (CEA) derived from liver metastases ofhuman colonic adenocarcinoma according to the procedure of Krupey et al.(Immunochem. 9: 617 (1972)), as modified by Newman et al. (Cancer Res.34:2125 (1974)). See Primus et al., Cancer Res. 43:686-92 (1983); U.S.Pat. No. 4,818,709.

Yet another suitable antibody is the NP-1 monoclonal antibody. The NP-1has similar specificity to that of MN-15. NP-1 was isolated fromhybridomas derived from BALB/c mice which were immunized with partiallypurified carcinoembryonic antigen (CEA) derived from liver metastases ofhuman colonic adenocarcinoma according to the procedure of Krupey et al.(Immunochem. 9:617 (1972)), as modified by Newman et al. (Cancer Res.34:2125 (1974)). See Primus et al., Cancer Res., 43:686-92 (1983); U.S.Pat. No. 4,818,709.

The MN3 antibodies, fusion proteins, and fragments thereof of thepresent invention may also be administered with another conjugated orunconjugated MN3 antibody, fusion protein, or fragment thereof, or aconjugated or unconjugated non-MN3 antibody, fusion protein, or fragmentthereof.

The chimeric or humanized MN3 MAbs and fragments thereof of the presentinvention contain specific murine CDRs or a combination of murine CDRsfrom more than one murine or chimeric MN3 MAb. Preferably, the chimericand humanized MN3 antibodies of the present invention contain CDRs froma murine MN3 antibody. The MN3 Mabs and fragments thereof of the presentinvention are murine, humanized, chimeric or fully human Mabs. Thechimeric and humanized antibodies contain the amino acid sequence of theCDRs of a murine MN3 (mMN3) MAb and the light and heavy chain constantregions of a human antibody.

In a preferred embodiment, the humanized MN3 MAb or fragment thereof ofthe present invention comprises the CDRs of a murine MN3 MAb and theframework (FR) regions of the light and heavy chain variable regions ofa human antibody and the light and heavy chain constant regions of ahuman antibody. Preferably, the CDRs of the light chain variable regionof the humanized MN3 MAb comprise a CDR1 that comprises an amino acidsequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 1), CDR2 that comprises anamino acid sequence of KVSNRFS (SEQ ID NO: 2), and/or CDR3 thatcomprises an amino acid sequence of FQGSHVPPT (SEQ ID NO: 3); and theCDRs of the heavy chain variable region of the MN3 MAb comprise a CDR1that comprises an amino acid sequence of NYGMN (SEQ ID NO: 4), a CDR2that comprises an amino acid sequence of WINTYTGEPTYADDFKG (SEQ ID NO:5), and/or a CDR3 that comprises an amino acid sequence of KGWMDFNGSSLDY(SEQ ID NO: 6).

In another embodiment, the humanized MN3 MAb or fragment thereof mayfurther contain in the FRs of the light and heavy chain variable regionsof the hMN3 antibody, at least one amino acid from the corresponding FRsof the murine MAb. In one embodiment, the humanized MN3 MAb or fragmentthereof contains at least one amino acid residue 27, 30, 67, 68, 69 or94 of the murine heavy chain variable region, for example as shown inthe figures, and/or of at least one amino acid residue 20, 22, 39, 60,70 or 100 of the murine light chain variable region, such as those shownin the figures. One or more of the murine amino acid sequences can bemaintained in the human FR regions of the light and heavy variablechains if necessary to maintain proper binding or to enhance binding tothe antigen recognized by MN3. More preferably the humanized MN3 MAb orfragment thereof of the present invention comprises the hMN3VH of FIG.4B and the hMN3VK of FIG. 4A.

In a related vein, chimeric MN3 (cMN3) MAb or fragment thereof of thepresent invention comprises the CDRs of a murine MN3 MAb and the FRregions of the light and heavy chain variable regions of the murine MN3MAb. In other words, the cMN3 antibody comprises the Fvs of the parentalmurine (i.e., mMN3) MAb, and the light and heavy chain constant regionsof a human antibody, wherein the CDRs of the light chain variable regionof the chimeric MN3 MAb comprise a CDR1 that comprises an amino acidsequence of RSSQSIVHSNGNTYLE (SEQ ID NO: 1), CDR2 that comprises anamino acid sequence of KVSNRFS (SEQ ID NO: 2), and/or CDR3 thatcomprises an amino acid sequence of FQGSHVPPT (SEQ ID NO: 3); and theCDRs of the heavy chain variable region of the MN3 MAb comprise a CDR1that comprises an amino acid sequence of NYGMN (SEQ ID NO: 4), a CDR2that comprises an amino acid sequence of WINTYTGEPTYADDFKG (SEQ ID NO:5), and/or a CDR3 that comprises an amino acid sequence of KGWMDFNGSSLDY(SEQ ID NO: 6).

More preferably the chimeric MN3 MAb or fragment thereof comprises thecomplementarity-determining regions (CDRs) of a murine MN3 MAb and theframework (FR) regions of the light and heavy chain variable regions ofthe murine MN3 MAb and the light and heavy chain constant regions of ahuman antibody, wherein the CDRs and FRs of the heavy and light chainvariable region of the chimeric MN3 MAb comprise the sequence shown inFIGS. 2B and 2A, respectively, designated cMN3VH and cMN3VK.

The present invention also contemplates antibody fusion proteins orfragments thereof comprising at least two MN3 MAbs or fragments thereof.Preferably, the MN3 antibodies and fragments thereof are the MN3antibodies and fragments thereof of the present invention. Alsopreferred, the antibody fusion proteins of the present invention arecomposed of one MN3 MAb and one or more of the second MAbs to providespecificity to different antigens, and are described in more detailbelow. In a preferred embodiment, the MN3 antibody is an MN3 antibody.The antibody fusion protein or fragment thereof of the present inventionis also intended to encompass an antibody fusion protein or fragmentthereof comprising at least one first MN3 MAb or fragment thereof asdescribed above and at least one second non-MN3 MAb or fragment thereof.Preferably, the non-MN3 antibody or fragment thereof is a granulocyteassociated antibody. A variety of anti-granulocyte antibodies can beused in the present invention. Examples include, but are not limited to,anti-NCA-90, anti-NCA-95, MN-2, MN-3, MN-15, NP-1, NP-2, BW 250/183, andMAb 47 and antibodies directed to antigens present on a singlegranulocyte precursor, such as anti-CD-15 and anti-CD-33.

The humanized, chimeric and human MN3 antibody may possess enhancedaffinity binding with the epitope as a result of CDR mutation andmanipulation of the CDR and other sequences in the variable region toobtain a superior therapeutic agent for the treatment of leukemia, andin particular myelogenous, or myeloid, leukemias. Modification to thebinding specificity, affinity or avidity of an antibody is known anddescribed in WO 98/44001, as affinity maturation, and this applicationsummarizes methods of modification and is incorporated in its entiretyby reference.

It may also be desirable to modify the antibodies of the presentinvention to improve effector function, e.g., so as to enhanceantigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC) of the antagonist. One or more amino acidsubstitutions or the introduction of cysteine in the Fc region may bemade, thereby improving internalization capability and/or increasedcomplement-mediated cell killing and ADCC. See Caron et al., J. Ex. Med.176:1191-1195 (1991) and Shopes, B. J. Immunol. 148:2918-2022 (1992),incorporated herein by reference in their entirety. An antibody fusionprotein may be prepared that has dual Fc regions with both enhancedcomplement lysis and ADCC capabilities.

Another embodiment of the present invention is a DNA sequence comprisinga nucleic acid encoding a MAb or fragment thereof selected from thegroup consisting of:

(a) an MN3 MAb or fragment thereof as described herein,

(b) an antibody fusion protein or fragment thereof comprising at leastof the MN3 MAbs or fragments thereof of the present invention,

(c) an antibody fusion protein or fragment thereof comprising at leastone first MAb or fragment thereof comprising an MN3 MAb or fragmentthereof as described herein and at least one second MAb or fragmentthereof, other than the MN3 MAb or fragment thereof described herein,and

(d) an antibody fusion protein or fragment thereof comprising at leastone first MAb or fragment thereof comprising the MN3 MAb or fragmentthereof and at least one second MAb or fragment thereof, wherein thesecond MAb is a an anti-granulocyte antibodies can be used in thepresent invention including, but not limited to, anti-NCA-90,anti-NCA-95, MN-2, MN-3, MN-15, NP-1, NP-2, BW 250/183, and MAb 47, aswell as antibodies against CD15 and CD33, and mixtures of all of theforgoing.

In a related vein, expression vectors comprising the DNA sequences arealso considered herein. In the case of vectors for use in preparing thehumanized, chimeric and human MN3 MAbs or antibody fusion proteinsthereof or fragments thereof, these vectors contain the coding sequencesfor the light and heavy chain constant regions and the hinge region ofthe human immunoglobulin, as well as the secretion signal peptide. Thesevectors additionally contain, where required, promoter/enhancer elementsto initiate the Ig gene expression in the selected host cell, and adrug-resistant marker for selection of transfected cells. Vectors thatare particularly useful in the present invention are DHFR (such aspdHL2) or GS-vector, particularly when used to express a chimeric,humanized or human antibody, such as an IgG, where the vector codes forthe heavy and light chain constant regions and hinge region of IgG1.More preferably, the light and heavy chain constant regions and hingeregion are from a human EU myeloma immunoglobulin, where optionally atleast one of the amino acid residues in the allotype positions ischanged to that found in a different IgG1 allotype, and whereinoptionally amino acid I253 of the heavy chain of EU (based on the EUnumbering system) may be replaced with alanine. See Edelman et al.,Proc. Natl. Acad Sci USA 63: 78-85 (1969), incorporated herein in itsentirety by reference.

Host cells containing the DNA sequences encoding the MN3 MAbs orfragments thereof or antibody fusion proteins or fragments thereof ofthe present invention or host cells containing the vectors that containthese DNA sequences are encompassed by the present invention.Particularly useful host cells are mammalian cells, and morespecifically, myeloma cell lines, such as Sp2/0, YB2/0, NS0, and CHO,such as DG-44, as discussed in more detail below. Also useful forproducing monoclonal antibodies and other fusion proteins is the PER.C6®human cell line.

Also encompassed by the present invention is the method of expressing aMN3 MAb or fragment thereof or a MN3 fusion protein or fragment thereofcomprising: (a) transfecting a mammalian cell with a DNA sequence ofencoding a MN3 MAb or fragment thereof or an antibody fusion protein orfragments thereof, and (b) culturing the cell transfected with the DNAsequence that secretes the MN3 or fragment thereof or MN3 antibodyfusion protein or fragment thereof. Known techniques may be used thatinclude a selection marker on the vector so that host cells that expressthe MAbs and the marker can be easily selected.

The present invention also encompasses liver cell targetingdiagnostic/detection or therapeutic immunoconjugates comprising an MN3MAb or fragment thereof or an MN3 fusion protein or fragment thereof,that bind to cell expressing the antigen recognized by MN3 and is boundto at least one diagnostic/detection and/or at least one therapeuticagent.

In a preferred embodiment, the diagnostic/detection immunoconjugatecomprises an MN3 MAb or fragment thereof or an antibody fusion proteinor fragment thereof, and at least one diagnostic/detection agent.Examples of diagnostic/detection agents include diverse labels,radionuclides, chelators, dyes, fluorescent compounds, chromagens, andother marker moieties. Radionuclides useful for positron emissiontomography include, but are not limited to: F-18, Mn-51, Mn-52m, Fe-52,Co-55, Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86,Zr-89, Tc-94m, In-110, I-120, and I-124. Total decay energies of usefulpositron-emitting radionuclides are preferably <2,000 keV, morepreferably under 1,000 keV, and most preferably <700 keV. Radionuclidesuseful as diagnostic agents utilizing gamma-ray detection include, butare not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75,Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197, andTl-201. Decay energies of useful gamma-ray emitting radionuclides arepreferably 20-2000 keV, more preferably 60-600 keV, and most preferably100-300 keV. The diagnostic agent of the present invention may also be acontrast agent such as manganese, iron or gadolinium.

Also preferred, the therapeutic immunoconjugate of the present inventioncomprises an MN3 antibody or fragment thereof, or an MN3 fusion proteinor fragment thereof, and at least one therapeutic agent. Examples oftherapeutic agents include a radioactive label, an immunomodulator, ahormone, a photoactive therapeutic agent, a cytotoxic agent, which maybe a drug or a toxin, and a combination thereof. The drugs useful in thepresent invention are those drugs that possess the pharmaceuticalproperty selected from the group consisting of antimitotic, alkylating,antimetabolite, antibiotic, alkaloid, antiangiogenic, apoptotic agentsand combinations thereof. More specifically, these drugs are selectedfrom the group consisting of nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, COX-2 inhibitors, pyrimidine analogs, purine analogs,antibiotics, enzymes, epipodophyllotoxins, platinum coordinationcomplexes, vinca alkaloids, substituted ureas, methyl hydrazinederivatives, adrenocortical suppressants, antagonists, endostatin,taxols, camptothecins, anthracyclines, taxanes, and their analogs, and acombination thereof. The toxins encompassed by the present invention arebacterial, plant, or animal toxins, such as those selected from thegroup consisting of ricin, abrin, alpha toxin, saporin, onconase, i.e.,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.

Suitable immunomodulators for the present invention include cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. More specificallylymphotoxins, including tumor necrosis factor (TNF), hematopoieticfactors, including interleukin (IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,IL-18, IL-21), colony stimulating factor, including granulocyte-colonystimulating factor (G-CSF) or granulocyte macrophage-colony stimulatingfactor (GM-CSF)), interferon, including interferons-α, -β or -γ, andstem cell growth factor, including designated “S1 factor.”

Particularly useful therapeutic immunoconjugates comprise one or moreradioactive labels that have an energy between 60 and 700 keV. Suchradioactive labels include, but ar not limited to ³²P, ³³P, ⁴⁷Sc, 59Fe,⁶⁴Cu, ⁶⁷Cu, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁰Y, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, ²¹²Pb, ²¹³Bi, ⁵⁸Co, ⁶⁷Ga,^(80m)Br, ^(99m)Tc, ^(103m)Rh, ¹⁰⁹Pt, ¹¹¹In, ¹¹⁹Sb, ¹²⁵I, ¹⁶¹Ho,^(189m)Os, ¹⁹²Ir, ¹⁵²Dy, ²¹¹At, ²¹²Bi, ²²³Ra, ²¹⁹Rn, ²¹⁵Po, ²¹¹Bi,²²⁵Ac, ²²¹Fr, ²¹⁷At, ²¹³Bi and ²⁵⁵Fm, and combinations thereof. Otheruseful therapeutic conjugates are photoactive therapeutic agent, such asa chromogen or dye.

The present invention particularly encompasses methods of treating,detecting or imaging sites of inflammation, including inflammationresulting from appendicitis, inflammatory bowel disease, pelvicinflammatory disease, fever of unknown origin and cystic fibrosis, waswell as in treating granulocyte related disorders, such as myeloidleukemia, in a subject, such as a mammal, including humans, domestic orcompanion pets, such as dogs and cats, comprising administering to thesubject a therapeutically effective amount of an MN3 MAb or a fragmentthereof of the present invention, formulated in a pharmaceuticallyacceptable vehicle. Therapeutic applications of Mabs are discussed inMagic bullets hit the target. Nature 417:584-586, 2002. Preferably theMN3 antibody or fragment thereof is an MN3 antibody or fragment thereof.This therapy utilizes a “naked antibody” that does not have atherapeutic agent bound to it. The administration of the “naked MN3antibody” can be supplemented by administering to the subjectconcurrently or sequentially a therapeutically effective amount of atleast one other “naked antibody” that binds to or is reactive withanother antigen on the surface of the target cell or that has otherfunctions, such as effector functions in the Fc portion of the MAb, thatis therapeutic and which is discussed herein. For example, preferredMAbs that can supplement the naked MN3 antibody are humanized, chimeric,human or murine (in the case of non-human animals) anti-granulocyteantibodies. Examples include, but are not limited to, anti-NCA-90,anti-NCA-95, MN-2, MN-3, MN-15, NP-1, NP-2, BW 250/183, and MAb 47, aswell as antibodies against CD15 and CD33.

Both the naked MN3 antibody therapy alone or in combination with othernaked MAbs or fragments thereof as discussed above can be furthersupplemented with the administration, either concurrently orsequentially, of a therapeutically effective amount of at least onetherapeutic agent, formulated in a pharmaceutically acceptable vehicle.As discussed herein the therapeutic agent may comprise a cytotoxicagent, a radioactive label, an immunomodulator, a hormone, a photoactivetherapeutic agent or a combination thereof, formulated in apharmaceutically acceptable vehicle.

In another therapeutic method, both the naked MN3 therapy alone or incombination with other naked MAbs, as discussed above, can be furthersupplemented with the administration, either concurrently orsequentially, of a therapeutically effective amount of at least onetherapeutic immunoconjugate, described herein and formulated in apharmaceutically acceptable vehicle. The therapeutic immunoconjugatecomprises at least one humanized, chimeric, human or murine (fornon-human subjects) MAb selected from the group consisting of a MAbsreactive with NCA-90, NCA-95, CD15, CD33, and from the MAbs MN2, MN3,MN-15, NP-1, NP-2, BW 250/183, and MAb 47. The therapeuticimmunoconjugate may be conjugated to at least one therapeutic agentselected from the group consisting of a cytotoxic agent, a radioactivelabel, an immunomodulator, a hormone, a photoactive therapeutic agent ora combination thereof, formulated in a pharmaceutically acceptablevehicle.

As described herein the methods provide methods of imaging sites ofinflammation, including inflammation resulting from appendicitis,inflammatory bowel disease, pelvic inflammatory disease, fever ofunknown origin and cystic fibrosis, was well as in treatinggranulocyte-related disorders, such as myeloid leukemia, in a subjectcomprising administering to a subject a therapeutically effective amountof an antibody fusion protein or fragment thereof comprising at leasttwo MN3 MAbs or fragments thereof of the present invention or comprisingat least one MN3 MAb or fragment thereof of the present invention and atleast one granulocyte associated MAb. Preferably, the anti-granulocyteantibody is elected from the group consisting of MAbs reactive withNCA-90, NCA-95, CD15, CD33, and from the MAbs MN2, MN3, MN-15, NP-1,NP-2, BW 250/183, and MAb 47.

These imaging and therapeutic methods can further be supplemented withthe administration to the subject concurrently or sequentially of atherapeutically effective amount of at least one therapeutic agent,formulated in a pharmaceutically acceptable vehicle, wherein thetherapeutic agent is preferably a cytotoxic agent, a radioactive label,an immunomodulator, a hormone, a photoactive therapeutic agent or acombination thereof, formulated in a pharmaceutically acceptablevehicle.

Further, the antibody fusion proteins and fragments thereof of thepresent invention can be administered to a subject concurrently orsequentially with a therapeutically effective amount of a therapeuticconjugate comprising at least one MAb bound to at least one therapeuticagent, wherein said MAb component of the conjugate preferably comprisesat least one humanized, chimeric, human or murine (for non-humansubjects) MAb selected from the group consisting of a MAbs reactive withNCA-90, NCA-95, CD15, CD33, and from the MAbs MN2, MN3, MN-15, NP-1,NP-2, BW 250/183, and MAb 47.

The antibody fusion protein itself may also be conjugated to at leastone therapeutic agent. These therapeutic agents can be a combination ofdifferent recited agents or combinations of the same agents, such as twodifferent therapeutic radioactive labels.

Also encompassed by the present invention is a method ofdiagnosing/detecting inflammation and granulocyte related disorders orcancers in a subject comprising administering to the subject, such as amammal, including humans and domestic and companion pets, such as dogs,cats, rabbits, guinea pigs, a diagnostic/detection immunoconjugatecomprising an MN3 MAb or fragment thereof or an MN3 fusion protein orfragment thereof of the present invention that binds to the cellexpressing an antigen recognized by MN3, wherein the MN3 MAb or fragmentthereof or antibody fusion protein or fragment thereof is bound to atleast one diagnostic/detection agent. Optionally, thediagnostic/detection immunoconjugate is formulated in a pharmaceuticallyacceptable vehicle. The useful diagnostic agents are described herein.

2. Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an MN3 monoclonal antibody fragment bindswith an epitope recognized by MN3. The term “antibody fragment” alsoincludes any synthetic or genetically engineered protein that acts likean antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), and minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion.

The term anti-granulocyte antibody refers to an antibody whichrecognizes an antigen which is present on one or more cell-types of theneutrophil/granulocyte/myelocyte lineage.

A naked antibody is generally an entire antibody which is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector functions, such as complementfixation and ADCC (antibody-dependent cell cytotoxicity), which setmechanisms into action that may result in cell lysis. Naked antibodiesinclude both polyclonal and monoclonal antibodies, as well as certainrecombinant antibodies, such as chimeric, humanized or human antibodies.However, it is possible that the Fc portion is not required fortherapeutic function, rather an antibody exerts its therapeutic effectthrough other mechanisms, such as induction of cell cycle resting andapoptosis. In this case, naked antibodies also include the unconjugatedantibody fragments defined above.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity-determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as acat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a subfragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents or dyes and radioisotopes.

A diagnostic agent is a molecule or atom which is administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing or detecting a disease bylocating the cells containing the antigen. Useful diagnostic agentsinclude, but are not limited to, radioisotopes, dyes (such as with thebiotin-streptavidin complex), contrast agents, fluorescent compounds ormolecules and enhancing agents (e.g., paramagnetic ions) for magneticresonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI techniqueand the preparation of antibodies conjugated to a MRI enhancing agentand is incorporated in its entirety by reference. Preferably, thediagnostic agents are selected from the group consisting ofradioisotopes, enhancing agents for use in magnetic resonance imaging,and fluorescent compounds. In order to load an antibody component withradioactive metals or paramagnetic ions, it may be necessary to react itwith a reagent having a long tail to which are attached a multiplicityof chelating groups for binding the ions. Such a tail can be a polymersuch as a polylysine, polysaccharide, or other derivatized orderivatizable chain having pendant groups to which can be boundchelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crownethers, bis-thiosemicarbazones, polyoximes, and like groups known to beuseful for this purpose. Chelates are coupled to the antibodies usingstandard chemistries. The chelate is normally linked to the antibody bya group which enables formation of a bond to the molecule with minimalloss of immunoreactivity and minimal aggregation and/or internalcross-linking other, more unusual, methods and reagents for conjugatingchelates to antibodies are disclosed in U.S. Pat. No. 4,824,659 toHawthorne, entitled “Antibody Conjugates,” issued Apr. 25, 1989, thedisclosure of which is incorporated herein in its entirety by reference.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I,¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N,¹⁵O, ⁷⁶Br, for radio-imaging. The same chelates, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI, when used along with the antibodies of the invention.Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with avariety of metals and radiometals, most particularly with radionuclidesof gallium, yttrium and copper, respectively. Such metal-chelatecomplexes can be made very stable by tailoring the ring size to themetal of interest. Other ring-type chelates such as macrocyclicpolyethers, which are of interest for stably binding nuclides, such as²²³Ra for RAIT are encompassed by the invention.

An immunoconjugate is a conjugate of an antibody component with atherapeutic or diagnostic agent. The diagnostic agent can comprise aradioactive or non-radioactive label, a contrast agent (such as formagnetic resonance imaging, computed tomography or ultrasound), and theradioactive label can be a gamma-, beta-, alpha-, Auger electron-, orpositron-emitting isotope.

An immunomodulator is a therapeutic agent as defined in the presentinvention that when present, typically stimulates immune cells toproliferate or become activated in an immune response cascade, such asmacrophages, B-cells, and/or T cells. An example of an immunomodulatoras described herein is a cytokine. As the skilled artisan willunderstand, certain interleukins and interferons are examples ofcytokines that stimulate T cell or other immune cell proliferation.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as an transgenicanimal, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell or cells. Suitablemammalian host cells include myeloma cells, such as SP2/0 cells, and NS0cells, as well as Chinese Hamster Ovary (CHO) cells, hybridoma celllines and other mammalian host cells useful for expressing antibodies.Also particularly useful to express MAbs and other fusion proteins, is ahuman cell line, PER.C6®, disclosed in WO 0063403 A2, which produces 2to 200-fold more recombinant protein as compared to conventionalmammalian cell lines, such as CHO, COS, Vero, Hela, BHK and SP2-celllines. Special transgenic animals with a modified immune system areparticularly useful for making fully human antibodies.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent natural antibody, single-chain antibody or antibody fragmentsegments with the same or different specificities are linked. An MN3fusion protein comprises a binding site for an antigen recognized byMN3. The MN3 fusion protein and fragment thereof of the presentinvention comprise at least one arm that binds to the same epitope anantibody or antibody fragment comprising CDR1 of a heavy chain variableregion that comprises an amino acid sequence of NYGMN, (SEQ ID NO: 4) aCDR2 that comprises an amino acid sequence of WINTYTGEPTYADDFKG (SEQ IDNO: 5), and/or a CDR3 that comprises an amino acid sequence ofKGWMDFNGSSLDY (SEQ ID NO: 6), and/or CDR1 of a light chain variableregion that comprises an amino acid sequence of RSSQSIVHSNGNTYLE (SEQ IDNO: 1), CDR2 that comprises an amino acid sequence of KVSNRFS (SEQ IDNO: 2), and/or CDR3 that comprises an amino acid sequence of FQGSHVPPT(SEQ ID NO: 3).

Valency of the fusion protein indicates the total number of binding armsor sites the fusion protein has to antigen(s) or epitope(s); i.e.,monovalent, bivalent, trivalent or multivalent. The multivalency of theantibody fusion protein means that it can take advantage of multipleinteractions in binding to an antigen, thus increasing the avidity ofbinding to the antigen, or to different antigens. Specificity indicateshow many different types of antigen or epitope an antibody fusionprotein is able to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., an IgGis bivalent because it has two binding arms but is monospecific becauseit binds to one type of antigen or epitope. A monospecific, multivalentfusion protein has more than one binding site for the same antigen orepitope. For example, a monospecific diabody is a fusion protein withtwo binding sites reactive with the same antigen. The fusion protein maycomprise a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise a therapeutic agent.Examples of therapeutic agents suitable for such fusion proteins includeimmunomodulators (“antibody-immunomodulator fusion protein”) and toxins(“antibody-toxin fusion protein”). One preferred toxin comprises aribonuclease (RNase), preferably a recombinant RNase.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and an antigen or epitope. One specificity would be for, forexample, a B-cell, T-cell, myeloid-, plasma-, or mast-cell antigen orepitope. Another specificity could be to a different antigen on the samecell type, such as CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, orCD22 on B-cells. Multispecific, multivalent antibodies are constructsthat have more than one binding site, and the binding sites are ofdifferent specificity. For example, a bispecific diabody, where onebinding site reacts with one antigen and the other with another antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, granulocyte or myeloid antigen orepitope and at least one other arm that specifically binds to atargetable conjugate that bears a therapeutic or diagnostic agent. Avariety of bispecific fusion proteins can be produced using molecularengineering. In one form, the bispecific fusion protein is divalent,consisting of, for example, a scFv with a single binding site for oneantigen and a Fab fragment with a single binding site for a secondantigen. In another form, the bispecific fusion protein is tetravalent,consisting of, for example, an IgG with two binding sites for oneantigen and two identical scFv for a second antigen.

Caninized or felinized antibodies are recombinant proteins in whichrodent (or another species) complementarity-determining regions of amonoclonal antibody (MAb) have been transferred from heavy and lightvariable chains of rodent (or another species) immunoglobulin into a dogor cat, respectively, immunoglobulin variable domain.

Domestic animals include large animals such as horses, cattle, sheep,goats, llamas, alpacas, and pigs, as well as companion animals. In apreferred embodiment, the domestic animal is a horse.

Companion animals include animals kept as pets. These are primarily dogsand cats, although small rodents, such as guinea pigs, hamsters, rats,and ferrets, are also included, as are subhuman primates such asmonkeys. In a preferred embodiment the companion animal is a dog or acat.

3. Preparation of Monoclonal Antibodies Including Chimeric, Humanizedand Human Antibodies

A monoclonal antibody (MAb) is an immunoglobulin that has specificbinding activity to a particular antigen and the antibody comprises onlyone antigen binding site that binds to only one epitope or one antigenicdeterminant of the antigen. Rodent monoclonal antibodies to specificantigens may be obtained by methods known to those skilled in the art.See, for example, Kohler and Milstein, Nature 256: 495 (1975), andColigan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter “Coligan”]. Briefly,monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Abs against peptides are generated by well-known methods for Abproduction. For example, injection of an immunogen, such as(peptide)_(n)-KLH, wherein KLH is keyhole limpet hemocyanin, and n=1-30,in complete Freund's adjuvant, followed by two subsequent injections ofthe same immunogen suspended in incomplete Freund's adjuvant intoimmunocompetent animals. The animals are given a final i.v. boost ofantigen, followed by spleen cell harvesting three days later. Harvestedspleen cells are then fused with Sp2/0-Ag14 myeloma cells and culturesupernatants of the resulting clones analyzed for anti-peptidereactivity using a direct-binding ELISA. Fine specificity of generatedAbs can be analyzed for by using peptide fragments of the originalimmunogen. These fragments can be prepared readily using an automatedpeptide synthesizer. For Ab production, enzyme-deficient hybridomas areisolated to enable selection of fused cell lines. This technique alsocan be used to raise antibodies to one or more of the chelatescomprising the linker, e.g., In(III)-DTPA chelates. Monoclonal mouseantibodies to an In(III)-di-DTPA are known (Barbet '395 supra).

After the initial raising of antibodies to the immunogen, the variablegenes of the monoclonal antibodies can be cloned from the hybridomacells, sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions with the murinecounterparts, which play an important role in antigen binding. In apreferred embodiment, one or more human residues in the frameworkregions of the humanized MN3 antibody or fragments thereof are replacedby their murine counterparts. It is also preferred that a combination offramework sequences from 2 different human antibodies are used forV_(H). Still preferred, the two human antibodies are EU and KOL. Theconstant domains of the antibody molecule are derived from those of ahuman antibody. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Techniques for constructing chimeric antibodies arewell known to those of skill in the art. As an example, Leung et al.,Hybridoma 13:469 (1994), describe how they produced an LL2 chimera bycombining DNA sequences encoding the V_(κ) and V_(H) domains of LL2monoclonal antibody, an anti-CD22 antibody, with respective human κ andIgG₁ constant region domains. This publication also provides thenucleotide sequences of the LL2 light and heavy chain variable regions,V_(κ) and V_(H), respectively. Techniques for producing humanized MAbsare described, for example, by Jones et al., Nature 321: 522 (1986),Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992),Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun.150: 2844 (1993), each of which is hereby incorporated by reference.

Another method for producing the antibodies of the present invention isby production in the milk of transgenic livestock. See, e.g., Colman,A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690,both of which are incorporated in their entirety by reference. Two DNAconstructs are prepared which contain, respectively, DNA segmentsencoding paired immunoglobulin heavy and light chains. The DNA segmentsare cloned into expression vectors which contain a promoter sequencethat is preferentially expressed in mammary epithelial cells. Examplesinclude, but are not limited to, promoters from rabbit, cow and sheepcasein genes, the cow α-lactoglobulin gene, the sheep β-lactoglobulingene and the mouse whey acid protein gene. Preferably, the insertedfragment is flanked on its 3′ side by cognate genomic sequences from amammary-specific gene. This provides a polyadenylation site andtranscript-stabilizing sequences. The expression cassettes arecoinjected into the pronuclei of fertilized, mammalian eggs, which arethen implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of bothtransgenes by Southern analysis. In order for the antibody to bepresent, both heavy and light chain genes must be expressed concurrentlyin the same cell. Milk from transgenic females is analyzed for thepresence and functionality of the antibody or antibody fragment usingstandard immunological methods known in the art. The antibody can bepurified from the milk using standard methods known in the art.

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody (MAb) can also be humanized by replacingthe sequences of the murine FR in the variable domains of the chimericMAb with one or more different human FRs. Specifically, mouse CDRs aretransferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239: 1534 (1988). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, WO0029584A1.

A fully human antibody of the present invention, i.e., a human MN3 MAbor another human antibody, used for combination therapy with humanizedor chimeric MN3 antibodies, can be obtained from a transgenic non-humananimal. See, e.g., Mendez et al., Nature Genetics, 15: 146-156 (1997)and U.S. Pat. No. 5,633,425, which are incorporated in their entirety byreference. For example, a human antibody can be recovered from atransgenic mouse possessing human immunoglobulin loci. The mouse humoralimmune system is humanized by inactivating the endogenous immunoglobulingenes and introducing human immunoglobulin loci. The humanimmunoglobulin loci are exceedingly complex and comprise a large numberof discrete segments which together occupy almost 0.2% of the humangenome. To ensure that transgenic mice are capable of producing adequaterepertoires of antibodies, large portions of human heavy- andlight-chain loci generally are introduced into the mouse genome. This isaccomplished in a stepwise process beginning with the formation of yeastartificial chromosomes (YACs) containing either human heavy- orlight-chain immunoglobulin loci in germline configuration. Since eachinsert is approximately 1 Mb in size, YAC construction requireshomologous recombination of overlapping fragments of the immunoglobulinloci. The two YACs, one containing the heavy-chain loci and onecontaining the light-chain loci, are introduced separately into mice viafusion of YAC-containing yeast spheroblasts with mouse embryonic stemcells. Embryonic stem cell clones are then microinjected into mouseblastocysts. Resulting chimeric males are screened for their ability totransmit the YAC through their germline and are bred with mice deficientin murine antibody production. Breeding the two transgenic strains, onecontaining the human heavy-chain loci and the other containing the humanlight-chain loci, creates progeny which produce human antibodies inresponse to immunization.

Unrearranged human immunoglobulin genes also can be introduced intomouse embryonic stem cells via microcell-mediated chromosome transfer(MMCT). See, e.g., Tomizuka et al., Nature Genetics, 16: 133 (1997). Inthis methodology microcells containing human chromosomes are fused withmouse embryonic stem cells. Transferred chromosomes are stably retained,and adult chimeras exhibit proper tissue-specific expression.

As an alternative, an antibody or antibody fragment of the presentinvention may be derived from human antibody fragments isolated from acombinatorial immunoglobulin library. See, e.g., Barbas et al., METHODS:A Companion to Methods in Enzymology 2: 119 (1991), and Winter et al.,Ann. Rev. Immunol. 12: 433 (1994), which are incorporated by reference.Many of the difficulties associated with generating monoclonalantibodies by B-cell immortalization can be overcome by engineering andexpressing antibody fragments in E. coli, using phage display. To ensurethe recovery of high affinity, monoclonal antibodies a combinatorialimmunoglobulin library must contain a large repertoire size. A typicalstrategy utilizes mRNA obtained from lymphocytes or spleen cells ofimmunized mice to synthesize cDNA using reverse transcriptase. Theheavy- and light-chain genes are amplified separately by PCR and ligatedinto phage cloning vectors. Two different libraries are produced, onecontaining the heavy-chain genes and one containing the light-chaingenes. Phage DNA is isolated from each library, and the heavy-andlight-chain sequences are ligated together and packaged to form acombinatorial library. Each phage contains a random pair of heavy- andlight-chain cDNAs and upon infection of E. coli directs the expressionof the antibody chains in infected cells. To identify an antibody thatrecognizes the antigen of interest, the phage library is plated, and theantibody molecules present in the plaques are transferred to filters.The filters are incubated with radioactively labeled antigen and thenwashed to remove excess unbound ligand. A radioactive spot on theautoradiogram identifies a plaque that contains an antibody that bindsthe antigen. Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtained, forexample, from STRATAGENE Cloning Systems (La Jolla, Calif.).

Further, recent methods for producing bispecific MAbs include engineeredrecombinant MAbs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bispecific fusion proteins canbe produced using molecular engineering. In one form, the bispecificfusion protein is monovalent, consisting of, for example, a scFv with asingle binding site for one antigen and a Fab fragment with a singlebinding site for a second antigen. In another form, the bispecificfusion protein is divalent, consisting of, for example, an IgG with twobinding sites for one antigen and two scFv with two binding sites for asecond antigen.

Bispecific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.Recombinant methods can be used to produce a variety of fusion proteins.For example a fusion protein comprising a Fab fragment derived from ahumanized monoclonal MN3 antibody and a scFv derived from a murineanti-diDTPA can be produced. A flexible linker, such as GGGS (SEQ ID NO:27) connects the scFv to the constant region of the heavy chain of theMN3 antibody. Alternatively, the scFv can be connected to the constantregion of the light chain of another humanized antibody. Appropriatelinker sequences necessary for the in-frame connection of the heavychain Fd to the scFv are introduced into the VL and VK domains throughPCR reactions. The DNA fragment encoding the scFv is then ligated into astaging vector containing a DNA sequence encoding the CH1 domain. Theresulting scFv-CH1 construct is excised and ligated into a vectorcontaining a DNA sequence encoding the VH region of an MN3 antibody. Theresulting vector can be used to transfect an appropriate host cell, suchas a mammalian cell for the expression of the bispecific fusion protein.

Preparation of Chimeric, Humanized and Human MN3 Antibodies

Cell lines and culture media used in the present invention include MN3producing hybridoma cells and Sp2/0-Ag14 myeloma cells (ATCC, Manassas,Va.). These cells may be cultured in Hybridoma serum-free media (HSFM)(life Technologies, Grand Island, N.Y.) supplemented with 10% fetalbovine serum (FBS) (Hyclone Laboratories, Logan, Utah) and antibiotics(complete media). Alternatively, they may be cultured in Dulbecco'smodified Eagle's Medium (DMEM) supplemented with 10% FCS (Gibco/BRL,Gaithersburg, Md.) containing 10% of FCS and 75 μg/ml gantamicin(complete HSFM) or, where indicated, in HSFM containing onlyantibiotics. Selection of the transfectomas may be carried out incomplete HSFM containing and appropriate cytocidal drug, such ashygromycin (hyg) and methotrexate (MTX). All cell lines are preferablymaintained at 37° C. in 5% CO₂.

Obtaining Vκ and VH Gene Segments

Isolation of the Vκ and V_(H) gene segments can be accomplished byseveral means that are well-known in the art. Two such means include,but are not limited to, PCR cloning and cDNA library screening.

PCR cloning techniques are well-known in the art. In brief, however, PCRcloning of Vκ and V_(H) gene fragments may be accomplished as follows.Total RNA may be isolated from a MN3 hybridoma cell line usingcommercially available kits such as the Fast Track RNA Isolation kit(Invitrogen, San Diego, Calif.). The first strand cDNA may then bereverse transcribed from RNA using a cDNA cycle kit (Invitrogen). Inthis process, 5 μg of total RNA is annealed to an oligo dT or randomhexamer primer, or a murine IgG CH1-specific primer or a murineCκ-specific primer. Examples of such primers include CH1B (5′-ACA GTCACT GAG CTG G-3′) (SEQ ID NO: 28) and CK3-BH1 (5′-GCC GGA TCC TGA CTGGAT GGT GGG AAG ATG GAT ACA-3′) (SEQ ID NO: 29), respectively. The firststrand cDNA may be used as templates to amplify the V_(H) and Vκsequences by PCR, as described by Orlandi et al. For the VK region, aprimer pair such as VK1BACK (5′-GAC ATT CAG CTG ACC CAG TCT CCA-3′) (SEQID NO: 30) and IgKC3′ (5′-CTC ACT GGA TGG TGG GAA GAT GGA TAC AGTTGG-3′) (SEQ ID NO: 31) may be used. For the V_(H) region, a primer pairsuch as VH1BACK (5′-AGG T(C/G)(A/C) A(A/G)C TGC AG(C/G) AGT C(A/T)GG-3′) (SEQ ID NO: 32) and CH1B may be used. After amplification, the Vκand V_(H) fragments may then be gel-purified and cloned into a cloningvector such as the TA cloning vector (Invitrogen) for sequence analysesby the dideoxytermination method. Sequences confirmed to be ofimmunoglobulin origin may then be used to construct chimeric Abexpression vectors using methods described by Leung et al. (Hybridoma,13:469 (1994)).

As a preferred alternative to isolating the Vκ and V_(H) gene segmentsby PCR cloning, cDNA library screening may be utilized. cDNA screeningmethods also are well known in the art. In brief, however, a cDNAlibrary may be constructed from the mRNA extracted from the murine MN3hybridoma cells in pSPORT vector (Life Technologies). The first strandcDNA may be synthesized by priming poly A RNA from MN3 hybridoma with anoligo dT primer-NotI adaptor (Life Technologies). After the secondstrand synthesis and attachment of SalI adaptors, the cDNA pool may besize fractionated through a cDNA size fractionation column. FractionatedcDNA may then be ligated to pSPORT vector and subsequently transformedinto Escherichia coli DH5α. A library may then be plated, transferred tofilters, and amplified.

Screening of the cDNA library may be accomplished by hybridization withlabeled probes specific for the heavy and light chains. For example[³²P]-labeled probes such as MUCH-1 (5′-AGA CTG CAG GAG AGC TGG GAA GGTGTG CAC-3′) (SEQ ID NO: 33) for heavy chain and MUCK-1 (5′-GAA GCA CACGAC TGA GGC ACC TCC AGA TGT-3′) (SEQ ID NO: 34) for light chain. Clonesthat are positive on a first screening may be transferred to duplicateplates and screened a second time with the same probes.

RNA isolation, cDNA synthesis, and amplification can be carried out asfollows. Total cell RNA can be prepared from a MN3 hybridoma cell line,using a total of about 10⁷ cells, according to Sambrook et al.,(Molecular Cloning: A Laboratory Manual, Second ed., Cold Spring HarborPress, 1989), which is incorporated by reference. First strand cDNA canbe reverse transcribed from total RNA conventionally, such as by usingthe SuperScript™ preamplification system (Gibco/BRL, Gaithersburg, Md.).Briefly, in a reaction volume of 20 μl, 50 ng of random hexamer primerscan be annealed to 5 μg of RNAs in the presence of 2 μl of 10× synthesisbuffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl₂, 1 mg/ml BSA],1 μl of 10 mM dNTP mix, 2 μl of 0.1 M DTT, and 200 units of SuperScript™reverse transcriptase. The elongation step is initially allowed toproceed at room temperature for 10 min followed by incubation at 42° C.for 50 min. The reaction can be terminated by heating the reactionmixture at 90° C. for 5 min.

Synthesizing and labeling the screening probes can be accomplished bywell-known means. Depending on the detection systems utilized, probelabeling will vary. Many kits for this purpose are commerciallyavailable. One method for 32-P labeling of oligonucleotides includes theuse of with [γ-³²P]ATP (Amersham Arlington Heights, Ill.) and T4polynucleotide kinase (New England Biolabs, Beverly, Mass.), followed bycolumn purification.

Preparation of a Chimeric MN3 Antibody

In a preferred embodiment, the MN3 antibody is a chimeric MN3 antibody(cMN3). The Vκ PCR products may be subcloned into a pBR327 based stagingvector (VKpBR) as described by Leung et al., Hybridoma, 13:469 (1994).The V_(H) PCR products may be subcloned into a similar pBluescript-basedstaging vector (VHpBS). The fragments containing the Vκ and V_(H)sequences, along with the promoter and signal peptide sequences, can beexcised from the staging vectors using HindIII and BamHI restrictionendonucleases. The Vκ fragments (about 600 bp) can be subcloned into amammalian expression vector (for example, pKh) conventionally. pKh is apSVhyg-based expression vector containing the genomic sequence of thehuman kappa constant region, an Ig enhancer, a kappa enhancer and thehyg-resistant gene. Similarly, the about 800 bp V_(H) fragments can besubcloned into pG1g, a pSVgpt-based expression vector carrying thegenomic sequence of the human IgG1 constant region, an Ig enhancer andthe xanthine-guanine phosphoribosyl transferase (gpt) gene. The twoplasmids may be co-transfected into mammalian cells, such as Sp2/0-Ag14cells, by electroporation and selected for hygromycin resistance. YB2/0cell can also be used. Shitara et al., J. Immunol. Methods 167:271(1994). Colonies surviving selection are expanded, and supernatantfluids monitored for production of cMN3 MAb by an ELISA method. Anantibody expression level of between 0.10 and 2.5 μg/ml can be expectedwith this system.

Alternately, the Vκ and VH expression cassettes can be assembled in themodified staging vectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI andXhoI/BamHI fragments, respectively, and subcloned into a singleexpression vector, such as pdHL2, as described by Gilles et al., J.Immunol. Methods 125:191 (1989), Losman et al., Clin. Cancer Res. 5:3101(1999) and in Losman et al., Cancer, 80:2660 (1997) for the expressionin Sp2/0-Ag14 cells. Another vector that is useful in the presentinvention is the GS-vector, as described in Barnes et al.,Cytotechnology 32:109-123 (2000), which is preferably expressed in theNS0 cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(II), Nr. 8, 870-880 (1998).

Preparation of a Humanized MN3 Antibody

In another preferred embodiment, the MN3 antibody is a humanized MN3antibody (hMN3) generated by CDR grafting. Once proper human Vframeworks are chosen based on the sequence homology and the sequencesfor the hMN3Vκ and V_(H) domains are designed, CDR engrafting can beaccomplished by gene synthesis. In most cases, the DNA encoding the Vκor VH domain will be approximately 350 bp long. One of the constructionstrategies is to divide a V gene into two halves, each of which can begenerated using a long synthetic DNA oligonucleotide (>140 bases) as thetemplate and two short flanking oligonucleotides (<50 bases) as theprimers in a PCR reaction. By taking advantage of codon degeneracy, aunique restriction site may easily be introduced, without changing theencoded amino acids, at regions close to the middle of the V gene DNAsequence. The short flanking oligonucleotide PCR primers can be designedwith the necessary restriction sites to facilitate subsequent assemblyof the full length humanized V genes from the PCR generated fragments.The Vκ DNA segment may be subcloned into a pBR327 based staging vector(VKpBR) as described by Leung et al., Hybridoma, 13:469 (1994). The VHsegment may be subcloned into a similar pBluescript-based staging vector(VHpBS).

The HindIII/BamHI fragment containing the Ig promoter, leader sequenceand the hMN3V_(H) sequence can be excised from the staging vector andsubcloned to the corresponding sites in a pSVgpt-based vector, pG1g,which contains the genomic sequence of the human IgG constant region, anIg enhancer and a gpt selection marker, forming the final expressionvector, hMN3pG1g. Similar strategies can be employed for theconstruction of the hMN3VK sequence. The restriction site chosen for theligation of the PCR products for the long oligonucleotides can be NsiIin this case.

The DNA sequence containing the Ig promoter, leader sequence and thehMN3 Vκ sequence can be excised from the staging vector VKpBR bytreatment with BamHI/HindIII, and can be subcloned into thecorresponding sites of a pSVhyg-based vector, pKh, which contains thegenomic sequence of human kappa chain constant regions, a hygromycinselection marker, an Ig and a kappa enhancer, forming the finalexpression vector, hMN3pKh.

Transfection, and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of hMN3pKh (light chain expressionvector) and 20 μg of hMN3pG1g (heavy chain expression vector) can beused for the transfection of 5×106 SP2/0 myeloma cells byelectroporation (BioRad, Richmond, Calif.) according to Co et al., J.Immunol., 148: 1149 (1992) which is incorporated by reference. Followingtransfection, cells may be grown in 96-well microtiter plates incomplete HSFM medium (GIBCO, Gaithersburg, Md.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 μg/ml of hygromycin. Colonies typically emerge 2-3weeks post-electroporation. The cultures can then be expanded forfurther analysis.

Alternately, the Vκ and VH expression cassettes can be assembled in themodified staging vectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI andXhoI/BamHI fragments, respectively, and subcloned into a singleexpression vector, such as pdHL2, as described by Gilles et al., J.Immunol. Methods 125:191 (1989), Losman et al., Clin. Cancer Res. 5:3101(1999) and in Losman et al., Cancer, 80:2660 (1997) for the expressionin Sp2/0-Ag14 cells. Another vector that is useful in the presentinvention is the GS vector, as described in Barnes et al.,Cytotechnology 32:109-123 (2000), which is preferably expressed in theNS0 cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(II), Nr. 8, 870-880 (1998).

Screening the Clones and Isolating Antibodies

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab′)₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch, West Grove, Pa.) are added to the wells, (100 μl ofantibody stock diluted×10⁴, supplemented with the unconjugated antibodyto a final concentration of 1.0 μg/ml). Following an incubation of 1 h,the plates are washed, typically three times. A reaction solution, [100μl, containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS], is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4 N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbencies at 280/260 nm. Peak fractions arepooled, dialyzed against PBS, and the antibody concentrated, forexample, with the Centricon™ 30 (Amicon, Beverly, Mass.). The antibodyconcentration is determined by ELISA, as before, and its concentrationadjusted to about 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), isconveniently added to the sample as preservative.

The affinity of a chimeric, humanized or human MN3 antibody may beevaluated using a direct binding assay or a competitive binding assay.

Modifying/Optimizing the Recombinant Antibodies

As humanization sometimes results in a reduction or even loss ofantibody affinity, additional modification might be required in order torestore the original affinity (See, for example, Tempest et al.,Bio/Technology 9: 266 (1991); Verhoeyen et al., Science 239: 1534(1988)), which are incorporated by reference. Knowing that cMN3 exhibitsa binding affinity comparable to that of its murine counterpart,defective designs, if any, in the original version of cMN3 can beidentified by mixing and matching the light and heavy chains of cMN3 tothose of the humanized version. Preferably, some human residues in theframework regions are replaced by their murine counterparts. Alsopreferred, a combination of framework sequences from 2 different humanantibodies, such as EU and KOL are used for V_(H). For example, FR1-3can come from EU and FR 4 from KOL.

Other modifications, such as Asn-linked glycosylation sites, can beintroduced into a chimerized, human, or humanized MN3 antibody byconventional oligonucleotide directed site-specific mutagenesis.Detailed protocols for oligonucleotide-directed mutagenesis and relatedtechniques for mutagenesis of cloned DNA are well known. For example,see Sambrook et al. and Ausubel et al. above.

For example, to introduce an Asn in position 18 of hMN3 Vκ (FIG. 4A),one may alter codon 18 from AGA for Arg to AAC for Asn. To accomplishthis, a single stranded DNA template containing the antibody light chainsequence is prepared from a suitable strain of E. coli (e.g., dut⁻,ung⁻) in order to obtain a single strand DNA molecule containing a smallnumber of uracils in place of thymidine. Such a DNA template can beobtained by M13 cloning or by in vitro transcription using a SP6promoter. See, for example, Ausubel et al., eds., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, NY, 1987. An oligonucleotidecontaining the mutated sequence is synthesized conventionally, annealedto the single-stranded template and the product treated with T4 DNApolymerase and T4 DNA ligase to produce a double-stranded DNA molecule.Transformation of wild type E. (dut⁺, ung⁺) cells with thedouble-stranded DNA provides an efficient recovery of mutated DNA.

Alternatively, an Asn-linked glycosylation site can be introduced intoan antibody light chain using an oligonucleotide containing the desiredmutation as the primer and DNA clones of the variable regions for the Vkchain, or by using RNA from cells that produce the antibody of interestas a template. Also see, Huse, in ANTIBODY ENGINEERING: A PRACTICALGUIDE, Boerrebaeck, ed., W. H. Freeman & Co., pp. 103-120, 1992.Site-directed mutagenesis can be performed, for example, using theTRANSFORMER™ kit (Clonetech, Palo Alto, Calif.) according to themanufacturer's instructions.

Alternatively, a glycosylation site can be introduced by synthesizing anantibody chain with mutually priming oligonucleotides, one suchcontaining the desired mutation. See, for example, Uhlmann, Gene 71: 29(1988); Wosnick et al., Gene 60: 115 (1988); Ausubel et al., above,which are incorporated by reference.

Although the general description above referred to the introduction ofan Asn glycosylation site in position 18 of the light chain of anantibody, it will occur to the skilled artisan that it is possible tointroduce Asn-linked glycosylation sites elsewhere in the light chain,or even in the heavy chain variable region.

4. Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, Fab, Fv, sFv and the like. Otherantibody fragments include, but are not limited to: the F(ab)′₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab′ fragments, which can be generated by reducingdisulfide bridges of the F(ab)′₂ fragments. Alternatively, Fab′expression expression libraries can be constructed (Huse et al., 1989,Science, 246:1274-1281) to allow rapid and easy identification ofmonoclonal Fab′ fragments with the desired specificity. The presentinvention encompasses antibodies and antibody fragments.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,“Single Chain Antibody Variable Regions,” TIBTECH, Vol 9: 132-137(1991). These references are incorporated herein by reference.

To obtain high-affinity scFv, an scFv library with a large repertoirecan be constructed by isolating V-genes from non-immunized human donorsusing PCR primers corresponding to all known V_(H), V_(κ) and V_(λ) genefamilies. See, e.g., Vaughn et al., Nat. Biotechnol., 14: 309-314(1996). Following amplification, the V_(κ) and V_(λ) pools are combinedto form one pool. These fragments are ligated into a phagemid vector.The scFv linker, (Gly-Gly-Gly-Gly-Ser)₃, (SEQ ID NO: 35) is then ligatedinto the phagemid upstream of the V_(L) fragment. The V_(H) andlinker-V_(L) fragments are amplified and assembled on the J_(H) region.The resulting V_(H)-linker-V_(L) fragments are ligated into a phagemidvector. The phagemid library can be panned using filters, as describedabove, or using immunotubes (Nunc; Maxisorp). Similar results can beachieved by constructing a combinatorial immunoglobulin library fromlymphocytes or spleen cells of immunized rabbits and by expressing thescFv constructs in P. pastoris. See, e.g., Ridder et al., Biotechnology,13: 255-260 (1995). Additionally, following isolation of an appropriatescFv, antibody fragments with higher binding affinities and slowerdissociation rates can be obtained through affinity maturation processessuch as CDR3 mutagenesis and chain shuffling. See, e.g., Jackson et al.,Br. J. Cancer, 78: 181-188 (1998); Osbourn et al., Immunotechnology, 2:181-196 (1996).

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 100 Kd fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 50 Kd Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

5. Fusion Proteins

The antibody fusion proteins of the present invention comprise two ormore antibodies or fragments thereof and each of the antibodies thatcompose this fusion protein can contain a therapeutic agent ordiagnostic agent. In other words, the antibody fusion protein orfragment thereof can comprise at least one first MN3 MAb or fragmentthereof and at least one second MAb or fragment thereof that is not anMN3 MAb. In a preferred embodiment, the MN3 antibody or fragment thereofis an MN3 antibody or fragment thereof. Preferably, the second MAb is agranulocyte associated antibody, such as an antibody against MAbsreactive with NCA-90, NCA-95, CD15, CD33, and from the MAbs MN2, MN3,MN-15, NP-1, NP-2, BW 250/183, and MAb 47, or a combination thereof.

Additionally, one or more of the antibodies or fragments thereof thatcomprise the antibody fusion protein can have at least one therapeuticor diagnostic/detection agent attached. Further, thediagnostic/detection agents or therapeutic agents need not be the samebut can be different therapeutic agents; for example, one can attach adrug and a radioisotope to the same fusion protein. Particulary, an IgGcan be radiolabeled with ¹³¹I and attached to a drug. The ¹³¹I can beincorporated into the tyrosine of the IgG and the drug attached to theepsilon amino group of the IgG lysines. Both therapeutic and diagnosticagents also can be attached to reduced SH groups and to the carbohydrateside chains.

Also preferred, the antibody fusion protein of the present inventioncomprises at least two MN3 monoclonal antibodies or fragments thereof,and these may be to different epitopes of a granulocyte antigen, such asthose recognized by MN3, or of different human immunoglobulin backbonesequences (or IgGs). Preferably, the antibodies or fragments there ofare MN3 antibodies or fragments thereof.

Multispecific and Multivalent Antibodies

The MN3 antibodies and fragments thereof of the present invention, aswell as other antibodies with different specificities for use incombination therapy, can be made as a multispecific antibody, comprisingat least one binding site to an antigen recognized by MN3 and at leastone binding site to another antigen, or a multivalent antibodycomprising multiple binding sites to the same epitope or antigen. In apreferred embodiment, the multispecific antibody or fragment thereofcomprises at least one binding site to an MN3 epitope and at least onebinding site that is not to an antigen recognized by MN3. Alsopreferred, the multispecific antibody or fragment thereof comprises atleast one binding site to an MN3 epitope and at least one binding siteto a different epitope on an antigen recognized by MN3.

The present invention provides a bispecific antibody or antibodyfragment having at least one binding region that specifically binds anantigen recognized by MN3 and at least one other binding region thatspecifically binds another targeted cell marker or a targetableconjugate. The targetable conjugate comprises a carrier portion whichcomprises or bears at least one epitope recognized by at least onebinding region of the bispecific antibody or antibody fragment.Preferably, the bispecific antibody binds to an MN3 epitope.

A variety of recombinant methods can be used to produce bi-specificantibodies and antibody fragments. For example, bi-specific antibodiesand antibody fragments can be produced in the milk of transgeniclivestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147,1998; U.S. Pat. No. 5,827,690. Two DNA constructs are prepared whichcontain, respectively, DNA segments encoding paired immunoglobulin heavyand light chains. The fragments are cloned into expression vectors whichcontain a promoter sequence that is preferentially expressed in mammaryepithelial cells. Examples include, but are not limited to, promotersfrom rabbit, cow and sheep casein genes, the cow α-lactoglobulin gene,the sheep β-lactoglobulin gene and the mouse whey acid protein gene.Preferably, the inserted fragment is flanked on its 3′ side by cognategenomic sequences from a mammary-specific gene. This provides apolyadenylation site and transcript-stabilizing sequences. Theexpression cassettes are coinjected into the pronuclei of fertilized,mammalian eggs, which are then implanted into the uterus of a recipientfemale and allowed to gestate. After birth, the progeny are screened forthe presence of both transgenes by Southern analysis. In order for theantibody to be present, both heavy and light chain genes must beexpressed concurrently in the same cell. Milk from transgenic females isanalyzed for the presence and functionality of the antibody or antibodyfragment using standard immunological methods known in the art. Theantibody can be purified from the milk using standard methods known inthe art.

Other recent methods for producing bsAbs include engineered recombinantAbs which have additional cysteine residues so that they crosslink morestrongly than the more common immunoglobulin isotypes. See, e.g.,FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997. Another approachis to engineer recombinant fusion proteins linking two or more differentsingle-chain antibody or antibody fragment segments with the needed dualspecificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163,1997. A variety of bi-specific fusion proteins can be produced usingmolecular engineering. In one form, the bi-specific fusion protein ismonovalent, consisting of, for example, a scFv with a single bindingsite for one antigen and a Fab fragment with a single binding site for asecond antigen. In another form, the bi-specific fusion protein isdivalent, consisting of, for example, an IgG with two binding sites forone antigen and two scFv with two binding sites for a second antigen.

An MN3 multivalent antibody or fragment thereof is also contemplated inthe present invention. Preferably, the MN3 multivalent antibody orfragment thereof is a humanized MN3 multivalent antibody or fragmentthereof. This multivalent antibody is constructed by association of afirst and a second polypeptide. The first polypeptide comprises a firstsingle chain Fv molecule covalently linked to a firstimmunoglobulin-like domain which preferably is an immunoglobulin lightchain variable region domain. The second polypeptide comprises a secondsingle chain Fv molecule covalently linked to a secondimmunoglobulin-like domain which preferably is an immunoglobulin heavychain variable region domain. Each of the first and second single chainFv molecules forms a target binding site, and the first and secondimmunoglobulin-like domains associate to form a third target bindingsite.

A single chain Fv molecule with the VL-L-VH configuration, wherein L isa linker, may associate with another single chain Fv molecule with theVH-L-VL configuration to form a bivalent dimer. In this case, the VLdomain of the first scFv and the VH domain of the second scFv moleculeassociate to form one target binding site, while the VH domain of thefirst scFv and the VL domain of the second scFv associate to form theother target binding site.

Another embodiment of the present invention is an MN3 bispecific,trivalent antibody comprising two heterologous polypeptide chainsassociated non-covalently to form three binding sites, two of which haveaffinity for one target and a third which has affinity for a hapten thatcan be made and attached to a carrier for a diagnostic and/ortherapeutic agent. Preferably, the antibody has two MN3 binding sitesand one other binding site. The bispecific, trivalent targeting agentshave two different scFvs, one scFv contains two V_(H) domains from oneantibody connected by a short linker to the V_(L) domain of anotherantibody and the second scFv contains two V_(L) domains from the firstantibody connected by a short linker to the V_(H) domain of the otherantibody. The methods for generating multivalent, multispecific agentsfrom V_(H) and V_(L) domains provide that individual chains synthesizedfrom a DNA plasmid in a host organism are composed entirely of V_(H)domains (the V_(H)-chain) or entirely of V_(L) domains (the V_(L)-chain)in such a way that any agent of multivalency and multispecificity can beproduced by non-covalent association of one V_(H)-chain with oneV_(L)-chain. For example, forming a trivalent, trispecific agent, theV_(H)-chain will consist of the amino acid sequences of three V_(H)domains, each from an antibody of different specificity, joined bypeptide linkers of variable lengths, and the V_(L)-chain will consist ofcomplementary V_(L) domains, joined by peptide linkers similar to thoseused for the V_(H)-chain. Since the V_(H) and V_(L) domains ofantibodies associate in an anti-parallel fashion, the preferred methodin this invention has the V_(L) domains in the V_(L)-chain arranged inthe reverse order of the V_(H) domains in the V_(H)-chain.

Diabodies, Triabodies and Tetrabodies

The MN3 antibodies and fragments thereof of the present invention canalso be used to prepare functional bispecific single-chain antibodies(bscAb), also called diabodies, and can be produced in mammalian cellsusing recombinant methods. See, e.g., Mack et al., Proc. Natl. Acad.Sci., 92: 7021-7025, 1995, incorporated. For example, bscAb are producedby joining two single-chain Fv fragments via a glycine-serine linkerusing recombinant methods. The V light-chain (V_(L)) and V heavy-chain(V_(H)) domains of two antibodies of interest are isolated usingstandard PCR methods. The V_(L) and V_(H) cDNA's obtained from eachhybridoma are then joined to form a single-chain fragment in a two-stepfusion PCR. The first PCR step introduces the (Gly₄-Ser₁)₃ linker, (SEQID NO: 35) and the second step joins the V_(L) and V_(H) amplicons. Eachsingle chain molecule is then cloned into a bacterial expression vector.Following amplification, one of the single-chain molecules is excisedand sub-cloned into the other vector, containing the second single-chainmolecule of interest. The resulting bscAb fragment is subcloned into aneukaryotic expression vector. Functional protein expression can beobtained by transfecting the vector into chinese hamster ovary cells.Bispecific fusion proteins are prepared in a similar manner. Bispecificsingle-chain antibodies and bispecific fusion proteins are includedwithin the scope of the present invention.

For example, a humanized, chimeric or human or murine MN3 monoclonalantibody can be used to produce antigen specific diabodies, triabodies,and tetrabodies. The monospecific diabodies, triabodies, and tetrabodiesbind selectively to targeted antigens and as the number of binding siteson the molecule increases, the affinity for the target cell increasesand a longer residence time is observed at the desired location. Fordiabodies, the two chains comprising the V_(H) polypeptide of thehumanized MN3 MAb connected to the V_(K) polypeptide of the humanizedMN3 MAb by a five amino acid residue linker are utilized. Each chainforms one half of the humanized MN3 diabody. In the case of triabodies,the three chains comprising V_(H) polypeptide of the humanized MN3 MAbconnected to the V_(K) polypeptide of the humanized MN3 MAb by no linkerare utilized. Each chain forms one third of the hMN3 triabody.

Also contemplated in the present invention is a bi-specific antibody orantibody fragment having at least one arm that is reactive against atargeted tissue or cell, such as granulocytes, and at least one otherarm that is reactive against a targetable construct. Preferably, one armof the bispecific antibody binds an antigen recognized by MN3. Thetargetable construct is comprised of a carrier portion and at least 2units of a recognizable hapten. Examples of recognizable haptensinclude, but are not limited to, histamine succinoyl glycine (HSG) andfluorescein isothiocyanate. The targetable construct may be conjugatedto a variety of agents useful for treating or identifying diseasedtissue. The targetable construct can be of diverse structure, but isselected not only to avoid eliciting an immune responses, but also forrapid in vivo clearance when used within the bsAb targeting method.Hydrophobic agents are best at eliciting strong immune responses,whereas hydrophilic agents are preferred for rapid in vivo clearance;thus, a balance between hydrophobic and hydrophilic needs to beestablished. This is accomplished, in part, by relying on the use ofhydrophilic chelating agents to offset the inherent hydrophobicity ofmany organic moieties. Also, subunits of the targetable construct may bechosen which have opposite solution properties, for example, peptides,which contain amino acids, some of which are hydrophobic and some ofwhich are hydrophilic. Aside from peptides, carbohydrates may be used.

Large quantities of bscAb and fusion proteins can be produced usingEscherichia coli expression systems. See, e.g., Zhenping et al.,Biotechnology, 14: 192-196, 1996. A functional bscAb can be produced bythe coexpression in E. coli of two “cross-over” scFv fragments in whichthe V_(L) and V_(H) domains for the two fragments are present ondifferent polypeptide chains. The V light-chain (V_(L)) and Vheavy-chain (V_(H)) domains of two antibodies of interest are isolatedusing standard PCR methods. The cDNA's are then ligated into a bacterialexpression vector such that C-terminus of the V_(L) domain of the firstantibody of interest is ligated via a linker to the N-terminus of theV_(H) domain of the second antibody. Similarly, the C-terminus of theV_(L) domain of the second antibody of interest is ligated via a linkerto the N-terminus of the V_(H) domain of the first antibody. Theresulting dicistronic operon is placed under transcriptional control ofa strong promoter, e.g., the E. coli alkaline phosphatase promoter whichis inducible by phosphate starvation. Alternatively, single-chain fusionconstructs have successfully been expressed in E. coli using the lacpromoter and a medium consisting of 2% glycine and 1% Triton X-100. See,e.g., Yang et al., Appl. Environ. Microbiol., 64: 2869-2874, 1998. An E.coli, heat-stable, enterotoxin II signal sequence is used to direct thepeptides to the periplasmic space. After secretion, the two peptidechains associate to form a non-covalent heterodimer which possesses bothantigen binding specificities. The bscAb is purified using standardprocedures known in the art, e.g., Staphylococcal protein Achromatography.

Functional bscAbs and fusion proteins also can be produced in the milkof transgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63:141-147, 1998; U.S. Pat. No. 5,827,690. The bscAb fragment, obtained asdescribed above, is cloned into an expression vector containing apromoter sequence that is preferentially expressed in mammary epithelialcells. Examples include, but are not limited to, promoters from rabbit,cow and sheep casein genes, the cow α-lactoglobulin gene, the sheepβ-lactoglobulin gene and the mouse whey acid protein gene. Preferably,the inserted bscAb is flanked on its 3′ side by cognate genomicsequences from a mammary-specific gene. This provides a polyadenylationsite and transcript-stabilizing sequences. The expression cassette isthen injected into the pronuclei of fertilized, mammalian eggs, whichare then implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of theintroduced DNA by Southern analysis. Milk from transgenic females isanalyzed for the presence and functionality of the bscAb using standardimmunological methods known in the art. The bscAb can be purified fromthe milk using standard methods known in the art. Transgenic productionof bscAb in milk provides an efficient method for obtaining largequantities of bscAb.

Functional bscAb and fusion proteins also can be produced in transgenicplants. See, e.g., Fiedler et al., Biotech., 13: 1090-1093, 1995;Fiedler et al., Immunotechnology, 3: 205-216, 1997. Such productionoffers several advantages including low cost, large scale output andstable, long term storage. The bscAb fragment, obtained as describedabove, is cloned into an expression vector containing a promotersequence and encoding a signal peptide sequence, to direct the proteinto the endoplasmic reticulum. A variety of promoters can be utilized,allowing the practitioner to direct the expression product to particularlocations within the plant. For example, ubiquitous expression intobacco plants can be achieved by using the strong cauliflower mosaicvirus 35S promoter, while organ specific expression is achieved via theseed specific legumin B4 promoter. The expression cassette istransformed according to standard methods known in the art.Transformation is verified by Southern analysis. Transgenic plants areanalyzed for the presence and functionality of the bscAb using standardimmunological methods known in the art. The bscAb can be purified fromthe plant tissues using standard methods known in the art.

Additionally, transgenic plants facilitate long term storage of bscAband fusion proteins. Functionally active scFv proteins have beenextracted from tobacco leaves after a week of storage at roomtemperature. Similarly, transgenic tobacco seeds stored for 1 year atroom temperature show no loss of scFv protein or its antigen bindingactivity.

Functional bscAb and fusion proteins also can be produced in insectcells. See, e.g., Mahiouz et al., J. Immunol. Methods, 212: 149-160(1998). Insect-based expression systems provide a means of producinglarge quantities of homogenous and properly folded bscAb. Thebaculovirus is a widely used expression vector for insect cells and hasbeen successfully applied to recombinant antibody molecules. See, e.g.,Miller, L. K., Ann. Rev. Microbiol., 42: 177 (1988); Bei et al., J.Immunol. Methods, 186: 245 (1995). Alternatively, an inducibleexpression system can be utilized by generating a stable insect cellline containing the bscAb construct under the transcriptional control ofan inducible promoter. See, e.g., Mahiouz et al., J. Immunol. Methods,212: 149-160 (1998). The bscAb fragment, obtained as described above, iscloned into an expression vector containing the Drosphilametallothionein promoter and the human HLA-A2 leader sequence. Theconstruct is then transfected into D. melanogaster SC-2 cells.Expression is induced by exposing the cells to elevated amounts ofcopper, zinc or cadmium. The presence and functionality of the bscAb isdetermined using standard immunological methods known in the art.Purified bscAb is obtained using standard methods known in the art.

The ultimate use of the bispecific diabodies described herein is forpre-targeting MN3 positive tumors for subsequent specific delivery ofdiagnostic/detection or therapeutic agents. These diabodies bindselectively to targeted antigens allowing for increased affinity and alonger residence time at the desired location. Moreover, non-antigenbound diabodies are cleared from the body quickly and exposure of normaltissues is minimized. The diagnostic/detection and therapeutic agentscan include isotopes, drugs, toxins, cytokines, hormones, growthfactors, conjugates, radionuclides, and metals. For example, gadoliniummetal is used for magnetic resonance imaging (MRI). Examples ofradionuclides are ²²⁵Ac, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶ Y, ¹¹¹In, ¹³¹I, ¹²⁵I,¹²³I, ^(99m)Tc, ^(94m)Tc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi,²¹³Bi, ³²P, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, and ²¹¹At. Other radionuclides are alsoavailable as diagnostic and therapeutic agents, especially those in theenergy range of 60 to 4,000 keV.

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius et al., Cancer Research(2000) 60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

7. MN3 Immunoconjugates

Any of the MN3 antibodies or fragments thereof, or antibody fusionproteins or fragments thereof of the present invention can be conjugatedwith one or more therapeutic and/or diagnostic/detection agents.Generally, one therapeutic or diagnostic/detection agent is attached toeach antibody or antibody fragment but more than one therapeutic agentor diagnostic agent can be attached to the same antibody, fusionprotein, or fragment thereof. Such a therapeutic or diagnostic/detectionagent may be a peptide which bears a diagnostic/detection or therapeuticagent. An immunoconjugate retains the immunoreactivity of the antibodycomponent, i.e., the antibody moiety has about the same or slightlyreduced ability to bind the cognate antigen after conjugation as beforeconjugation.

A wide variety of diagnostic/detection and therapeutic agents can beadvantageously conjugated to the antibody, fusion protein, or fragmentthereof of the present invention. In a preferred embodiment, thediagnostic/detection agents are selected from the group consisting ofradioisotopes for nuclear imaging, intraoperative and endoscopicdetection, enhancing agents for use in magnetic resonance imaging or inultrasonography, radiopaque and contrast agents for X-rays and computedtomography, and fluorescent compounds for fluoroscopy, includingendoscopic fluoroscopy. Fluorescent and radioactive agents conjugated toantibodies or used in bispecific, pretargeting methods, are particularlyuseful for endoscopic, intraoperative or intravascular detection of thetargeted antigens associated with diseased tissues or clusters of cells,such as malignant tumors, as disclosed in Goldenberg U.S. Pat. Nos.5,716,595, 6,096,289 and U.S. application Ser. No. 09/348,818,incorporated herein by reference in their entirety, particularly withgamma-, beta-, and positron-emitters. Radionuclides useful for positronemission tomography include, but are not limited to: F-18, Mn-51,Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m,Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120, and I-124.

The therapeutic agents recited here are those agents that also areuseful for administration separately with a naked antibody, as describedherein. Therapeutic agents include, for example, chemotherapeutic drugssuch as vinca alkaloids and other alkaloids, anthracyclines,epidophyllotoxins, taxanes, antimetabolites, alkylating agents,antibiotics, COX-2 inhibitors, antimitotics, antiangiogenic andapoptotoic agents, particularly doxorubicin, methotrexate, taxol,CPT-11, camptothecans, and others from these and other classes ofanticancer agents, and the like. Other useful cancer chemotherapeuticdrugs for the preparation of immunoconjugates and antibody fusionproteins include nitrogen mustards, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, pyrimidine analogs, purine analogs,platinum coordination complexes, hormones, toxins (e.g., RNAse,Pseudomonas exotoxin), and the like. Suitable chemotherapeutic agentsare described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an immunoconjugate of the MN3 antibodyor fragment thereof of the present invention. Additionally, the toxinmay be used in combination with a naked chimeric, humanized or human MN3antibody or fragment thereof, an MN3 fusion protein or fragment thereof,or a MN3 antibody or fragment thereof conjugated to a differenttherapeutic agent. Other toxins suitably employed in the preparation ofsuch conjugates or other fusion proteins, include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), and Goldenberg, CA—A Cancer Journal for Clinicians 44:43 (1994).Additional toxins suitable for use in the present invention are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499,which is incorporated in its entirety by reference. These can bederived, for example, from animal, plant and microbial sources, orchemically or recombinantly engineered. The toxin can be a plant,microbial, or animal toxin, or a synthetic variation thereof.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of the MN3 immunoconjugate, or beadministered unconjugated to the chimeric, humanized or human MN3antibody, fusion protein, or fragment thereof of the present invention.As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferons(e.g., interferons-α, -β and -γ), the stem cell growth factor designated“S1 factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21,interferon-γ, TNF-α, and the like. Alternatively, subjects can receive anaked MN3 antibody or fragment thereof, or naked fusion protein orfragment thereof, and a separately administered cytokine, which can beadministered before, concurrently or after administration of the nakedMN3 antibody or fragment, or naked MN3 fusion protein or fragmentthereof. The MN3 antibody or fragment there or fusion protein orfragment thereof of may also be conjugated to an immunomodulator. Theimmunomodulator may also be conjugated to a hybrid antibody consistingof one or more antibodies or antibody fragments binding to differentantigens. Such an antigen may also be an immunomodulator. For example,CD40 or other immunomodulators may be administered in combination with aMN3 antibody or fragment thereof either together, before or after theantibody combinations are administered.

Furthermore, an MN3 antibody or fragment thereof, or fusion protein orfragment thereof may comprise a γ-emitting radionuclide or apositron-emitter useful for diagnostic imaging. Examples ofdiagnostic/detection agents include diverse labels, radionuclides,chelators, dyes, contrast agents, fluorescent compounds, chromagens, andother marker moieties. Radionuclides useful for positron emissiontomography include, but are not limited to: ¹⁸F, ⁵¹Mn, ^(52m)Mn, ⁵²Fe,⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁸Ga, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, ⁸⁶Y, ⁸⁹Zr,^(94m)Tc, ¹¹⁰In, ¹²⁰I, and ¹²⁴I. Total decay energies of usefulpositron-emitting radionuclides are preferably <2,000 keV, morepreferably under 1,000 keV, and most preferably <700 keV. Radionuclidesuseful as diagnostic agents utilizing gamma-ray detection include, butare not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75,Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197, andTl-201. Decay energies of useful gamma-ray emitting radionuclides arepreferably 20-2000 keV, more preferably 60-600 keV, and most preferably100-300 keV.

Additionally, radionuclides suitable for treating a diseased tissueinclude, but are not limited to, P-32, P-33, Sc-47, Fe-59, Cu-64, Cu-67,Se-75, As-77, Sr-89, Y-90, Mo-99, Rh-105, Pd-109, Ag-111, I-125, I-131,Pr-142, Pr-143, Pm-149, Sm-153, Tb-161, Ho-166, Er-169, Lu-177, Re-186,Re-188, Re-189, Ir-194, Au-198, Au-199, Pb-211, Pb-212, and Bi-213,Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125,Ho-161, Os-189m, Ir-192, Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Fm-255 and combinations thereof.

Suitable diagnostic imaging isotopes are usually in the range of 20 to2,000 keV, while suitable therapeutic radionuclides are usually in therange of 20 to 10,000 keV. See for example, U.S. patent applicationentitled “Labeling Targeting Agents with Gallium-68”—Inventors G. L.Griffiths and W. J. McBride, (U.S. Provisional Application No.60/342,104), which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga,^(94m)Tc. and the like, for imaging purposes and which is incorporatedin its entirety by reference. A suitable radionuclide is an Augeremitter, and preferably has an energy of less than 1000 keV. Alsopreferred is a β emitter and has an energy between 20 and 5000 keV or analpha emitter and has an energy between 2000 and 10,000 keV.

A therapeutic or diagnostic/detection agent can be attached at the hingeregion of a reduced antibody component via disulfide bond formation. Asan alternative, such peptides can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same peptide that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

However, if the Fc region is absent, for example, if the antibody usedas the antibody component of the immunoconjugate is an antibodyfragment, it is still possible to attach a diagnostic/detection atherapeutic agent. A carbohydrate moiety can be introduced into thelight chain variable region of a full-length antibody or antibodyfragment. See, for example, Leung et al., J. Immunol. 154: 5919 (1995);Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat.No. 6,254,868, all of which are incorporated in their entirety byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

Targetable Constructs

The targetable construct can be of diverse structure, but is selectednot only to avoid eliciting an immune responses, but also for rapid invivo clearance when used within the bsAb targeting method. Hydrophobicagents are best at eliciting strong immune responses, whereashydrophilic agents are preferred for rapid in vivo clearance; thus, abalance between hydrophobic and hydrophilic needs to be established.This is accomplished, in part, by relying on the use of hydrophilicchelating agents to offset the inherent hydrophobicity of many organicmoieties. Also, subunits of the targetable construct may be chosen whichhave opposite solution properties, for example, peptides, which containamino acids, some of which are hydrophobic and some of which arehydrophilic. Aside from peptides, carbohydrates may be used.

Peptides having as few as two amino-acid residues may be used,preferably two to ten residues, if also coupled to other moieties suchas chelating agents. The linker should be a low molecular weightconjugate, preferably having a molecular weight of less than 50,000daltons, and advantageously less than about 20,000 daltons, 10,000daltons or 5,000 daltons, including the metal ions in the chelates. Forinstance, the known peptide DTPA-Tyr-Lys(DTPA)-OH (wherein DTPA isdiethylenetriaminepentaacetic acid) has been used to generate antibodiesagainst the indium-DTPA portion of the molecule. However, by use of thenon-indium-containing molecule, and appropriate screening steps, new Absagainst the tyrosyl-lysine dipeptide can be made. More usually, theantigenic peptide will have four or more residues, such as the peptideDOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (SEQ ID NO: 7), wherein DOTA is1,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the histaminesuccinoyl glycyl group of the formula:

The non-metal-containing peptide may be used as an immunogen, withresultant Abs screened for reactivity against the Phe-Lys-Tyr-Lys (SEQID NO: 36) backbone.

The invention also contemplates the incorporation of unnatural aminoacids, e.g., D-amino acids, into the backbone structure to ensure that,when used with the final bsAb/linker system, the arm of the bsAb whichrecognizes the linker moiety is completely specific. The inventionfurther contemplates other backbone structures such as those constructedfrom non-natural amino acids and peptoids. Examples of targetableconstructs that have D-amino acid backbones that can be used with thepresent methods include those disclosed in U.S. Patent Application No.60/478,403.

The peptides to be used as immunogens are synthesized conveniently on anautomated peptide synthesizer using a solid-phase support and standardtechniques of repetitive orthogonal deprotection and coupling. Freeamino groups in the peptide, that are to be used later for chelateconjugation, are advantageously blocked with standard protecting groupssuch as an acetyl group. Such protecting groups will be known to theskilled artisan. See Greene and Wuts Protective Groups in OrganicSynthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides areprepared for later use within the bsAb system, they are advantageouslycleaved from the resins to generate the corresponding C-terminal amides,in order to inhibit in vivo carboxypeptidase activity.

The haptens of the immunogen comprise an immunogenic recognition moiety,for example, a chemical hapten. Using a chemical hapten, preferably theHSG hapten, high specificity of the linker for the antibody isexhibited. This occurs because antibodies raised to the HSG hapten areknown and can be easily incorporated into the appropriate bispecificantibody. Thus, binding of the linker with the attached hapten would behighly specific for the antibody or antibody fragment.

Chelate Moieties

The presence of hydrophilic chelate moieties on the linker moietieshelps to ensure rapid in vivo clearance. In addition to hydrophilicity,chelators are chosen for their metal-binding properties, and are changedat will since, at least for those linkers whose bsAb epitope is part ofthe peptide or is a non-chelate chemical hapten, recognition of themetal-chelate complex is no longer an issue.

A chelator such as DTPA, DOTA, TETA, or NOTA or a suitable peptide, towhich a detectable label, such as a fluorescent molecule, or cytotoxicagent, such as a heavy metal or radionuclide, can be conjugated. Forexample, a therapeutically useful immunoconjugate can be obtained byconjugating a photoactive agent or dye to an antibody fusion protein.Fluorescent compositions, such as fluorochrome, and other chromogens, ordyes, such as porphyrins sensitive to visible light, have been used todetect and to treat lesions by directing the suitable light to thelesion. In therapy, this has been termed photoradiation, phototherapy,or photodynamic therapy (Jori et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain 22:430 (1986)). Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. Mew et al.,J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380 (1985);Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem.,Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin. Biol. Res.288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422 (1989); Pelegrinet al., Cancer 67:2529 (1991). However, these earlier studies did notinclude use of endoscopic therapy applications, especially with the useof antibody fragments or subfragments. Thus, the present inventioncontemplates the therapeutic use of immunoconjugates comprisingphotoactive agents or dyes.

Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with ⁴⁷Sc, ⁵²Fe, ⁵⁵Co, ⁶⁷Ga,⁶⁸Ga, ¹¹¹In, ⁸⁹Zr, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, and ²²⁵Ac forradio-imaging and RAIT. The same chelators, when complexed withnon-radioactive metals, such as Mn, Fe and Gd can be used for MRI, whenused along with the bsAbs of the invention. Macrocyclic chelators suchas NOTA (1,4,7-triaza-cyclononane-N,N′,N″-triacetic acid), DOTA, andTETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are ofuse with a variety of metals and radiometals, most particularly withradionuclides of Ga, Y and Cu, respectively.

DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group Ia and GroupIIIa metal cations. Such metal-chelate complexes can be made very stableby tailoring the ring size to the metal of interest. Other ring-typechelators such as macrocyclic polyethers are of interest for stablybinding nuclides such as ²²³Ra for RAIT. Porphyrin chelators may be usedwith numerous radiometals, and are also useful as certain cold metalcomplexes for bsAb-directed immuno-phototherapy. More than one type ofchelator may be conjugated to a carrier to bind multiple metal ions,e.g., cold ions, diagnostic radionuclides and/or therapeuticradionuclides. Particularly useful therapeutic radionuclides include,but are not limited to, ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag,¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac. Particularlyuseful diagnostic/detection radionuclides include, but are not limitedto, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd and ¹⁷⁵Lu.

Chelators such as those disclosed in U.S. Pat. No. 5,753,206, especiallythiosemi-carbazonylglyoxylcysteine (Tscg-Cys) andthiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators areadvantageously used to bind soft acid cations of Tc, Re, Bi and othertransition metals, lanthanides and actinides that are tightly bound tosoft base ligands, especially sulfur- or phosphorus-containing ligands.It can be useful to link more than one type of chelator to a peptide,e.g., a DTPA or similar chelator for, say In(III) cations, and athiol-containing chelator, e.g., Tscg-Cys, for Tc cations. Becauseantibodies to a di-DTPA hapten are known (Barbet '395, supra) and arereadily coupled to a targeting antibody to form a bsAb, it is possibleto use a peptide hapten with cold di-DTPA chelator and another chelatorfor binding a radioisotope, in a pretargeting protocol, for targetingthe radioisotope. One example of such a peptide isAc-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys-)-NH₂ (SEQ ID NO: 37). Thispeptide can be preloaded with In(III) and then labeled with 99-m-Tccations, the In(III) ions being preferentially chelated by the DTPA andthe Tc cations binding preferentially to the thiol-containing Tscg-Cys.Other hard acid chelators such as NOTA, DOTA, TETA and the like can besubstituted for the DTPA groups, and Mabs specific to them can beproduced using analogous techniques to those used to generate theanti-di-DTPA Mab.

It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the linker, e.g., with differentchelate ring sizes, to bind preferentially to two different hard acid orsoft acid cations, due to the differing sizes of the cations, thegeometries of the chelate rings and the preferred complex ion structuresof the cations. This will permit two different metals, one or both ofwhich may be radioactive or useful for MRI enhancement, to beincorporated into a linker for eventual capture by a pretargeted bsAb.

Preferred chelators include NOTA, DOTA and Tscg and combinationsthereof. These chelators have been incorporated into a chelator-peptideconjugate motif as exemplified as described herein, such as in thefollowing constructs:

-   (a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;-   (b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (SEQ ID NO: 7);-   (c) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

The chelator-peptide conjugates (d) and (e), above, has been shown tobind ⁶⁸Ga and is thus useful in positron emission tomography (PET)applications.

Chelators are coupled to the linker moieties using standard chemistrieswhich are discussed more fully in the working Examples below. Briefly,the synthesis of the peptideAc-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂ was accomplished by firstattaching Aloc-Lys(Fmoc)-OH to a Rink amide resin on the peptidesynthesizer. The protecting group abbreviations “Aloc” and “Fmoc” usedherein refer to the groups allyloxycarbonyl and fluorenylmethyloxycarbonyl. The Fmoc-Cys(Trt)-OH and TscG were then added to the sidechain of the lysine using standard Fmoc automated synthesis protocols toform the following peptide: Aloc-Lys(Tscg-Cys(Trt)-rink resin. The Alocgroup was then removed. The peptide synthesis was then continued on thesynthesizer to make the following peptide:(Lys(Aloc)-D-Tyr-Lys(Aloc)-Lys(Tscg-Cys(Trt)-)-rink resin. FollowingN-terminus acylation, and removal of the side chain Aloc protectinggroups. The resulting peptide was then treated with activatedN-trityl-HSG-OH until the resin gave a negative test for amines usingthe Kaiser test. See Karacay et al. Bioconjugate Chem. 11:842-854(2000). The synthesis of Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂,as well as the syntheses of DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; andDOTA-Phe-Lys(HSG)-Tyr -Lys(HSG)-NH₂ (SEQ ID NO: 7) are described ingreater detail below.

Preparation of Metal Chelates

Chelator-peptide conjugates may be stored for long periods as solids.They may be metered into unit doses for metal-binding reactions, andstored as unit doses either as solids, aqueous or semi-aqueoussolutions, frozen solutions or lyophilized preparations. They may belabeled by well-known procedures. Typically, a hard acid cation isintroduced as a solution of a convenient salt, and is taken up by thehard acid chelator and possibly by the soft acid chelator. However,later addition of soft acid cations leads to binding thereof by the softacid chelator, displacing any hard acid cations which may be chelatedtherein. For example, even in the presence of an excess of cold¹¹¹InCl₃, labeling with 99m-Tc(V) glucoheptonate or with Tc cationsgenerated in situ with stannous chloride and Na99m-TcO₄ proceedsquantitatively on the soft acid chelator. Other soft acid cations suchas ¹⁸⁶Re, ¹⁸⁸Re, ²¹³Bi and divalent or trivalent cations of Mn, Co, Ni,Pb, Cu, Cd, Au, Fe, Ag (monovalent), Zn and Hg, especially ⁶⁴Cu and⁶⁷Cu, and the like, some of which are useful for radioimmunodiagnosis orradioimmunotherapy, can be loaded onto the linker peptide by analogousmethods. Re cations also can be generated in situ from perrhenate andstannous ions or a prereduced rhenium glucoheptonate or othertranschelator can be used. Because reduction of perrhenate requires morestannous ion (typically above 200 μg/mL final concentration) than isneeded for the reduction of Tc, extra care needs to be taken to ensurethat the higher levels of stannous ion do not reduce sensitive disulfidebonds such as those present in disulfide-cyclized peptides. Duringradiolabeling with rhenium, similar procedures are used as are used withthe Tc-99m. A preferred method for the preparation of ReO metalcomplexes of the Tscg-Cys-ligands is by reacting the peptide withReOCl₃(P(Ph₃)₂ but it is also possible to use other reduced species suchas ReO(ethylenediamine)₂.

8. Humanized, Chimeric and Human Antibodies Use for Treatment andDiagnosis

Contemplated in the present invention is the use of murine, humanized,chimeric and human MN3 antibodies and fragments thereof in deliverymethods of therapeutic and diagnostic/detection agents, and therapeuticand diagnostic/detection methods. Preferably, the MN3 antibodies andfragments thereof are chimeric, humanized or human MN3 antibodies.

For example, a method of delivering a diagnostic/detection agent, atherapeutic agent, or a combination thereof to a target comprising (i)administering to a subject the antibody or fragment thereof an antibody,fusion protein, or fragment thereof; (ii) waiting a sufficient amount oftime for an amount of the non-binding protein to clear the subject'sblood stream; and (iii) administering to said subject a carrier moleculecomprising a diagnostic/detection agent, a therapeutic agent, or acombination thereof, that binds to a binding site of said antibody.Preferably, the carrier molecule binds to more than one binding site ofthe antibody.

The present invention also contemplates methods of diagnosing ordetecting a malignancy in a subject. Diagnosis/detection may beaccomplished by administering a diagnostically effective amount of adiagnostic/detection immunoconjugate, comprising an MN3 monoclonalantibody or fragment thereof or a fusion protein or fragment thereof,wherein said MN3 MAb or fragment thereof or fusion protein or fragmentthereof is bound to at least one diagnostic/detection agent, formulatedin a pharmaceutically acceptable excipient, and detecting said label.Preferably, the MN3 antibody, fusion protein, or fragment thereof is anMN3 antibody.

In a related vein, a method of diagnosing or detecting a malignancy in asubject comprising (i) performing an in vitro diagnosis assay on aspecimen from said subject with a composition comprising a MN3 MAb orfragment thereof or a antibody fusion protein or fragment thereof of anyone of the antibodies, fusion proteins, or fragments thereof of thepresent invention, is also considered. Preferably, the in vitrodiagnosis assay is selected from the group consisting of immunoassays,RT-PCR and immunohistochemistry.

In the methods described herein, radioactive and non-radioactive agentscan be used as diagnostic agents. A suitable non-radioactive diagnosticagent is a contrast agent suitable for magnetic resonance imaging, aradiopaque compound for X-rays or computed tomography, or a contrastagent suitable for ultrasound. Magnetic imaging agents include, forexample, non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, when used along with theantibodies of the invention. See U.S. Ser. No. 09/921,290 filed on Oct.10, 2001, which is incorporated in its entirety by reference. In apreferred embodiment, the contrast agent is an ultrasound-enhancingagent. Still preferred, the ultrasound-enhancing agent is a liposome.Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

Also described in the present invention is the use of murine, chimeric,humanized and human MN3 antibodies and fragments thereof in methods fortreating malignancies. For example, a malignancy of particular interestin this patent is a myeloid leukemia. The method comprises administeringto a subject a therapeutically effective amount of an antibody orfragment thereof or an antibody fusion protein or fragment thereofcomprising at least two MAbs or fragments thereof, wherein at least oneMN3 MAb or fragment thereof or fusion proteins or fragments thereof areany one of the antibodies of the present invention, formulated in apharmaceutically suitable excipient. In another embodiment, a secondMAb, fusion protein or fragment thereof is not an MN3 antibody, fusionprotein or fragment thereof.

In a related vein, a method of treating a cancer cell in a subjectcomprising (i) administering to said subject a therapeutically effectiveamount of a composition comprising a naked or conjugated MN3 MAb orfragment thereof or antibody fusion protein or fragment thereof, of anyone of the antibodies, fusion proteins, or fragments thereof of thepresent invention, (ii) formulating said MN3 MAb or fragment thereof orantibody fusion protein or fragment thereof in a pharmaceuticallysuitable excipient, is contemplated. Preferably, such a compositionfurther comprises a second antibody, fusion protein, or fragmentthereof. The second antibody, fusion protein, or fragment thereof can,but need not be an MN3 antibody, fusion protein or fragment thereof. Thepreferred mode of administration is parenterally. Examples of parentaladministration include through intravenous, subcutaneous, intramuscular,intradermal, intrathecal/intraspinal routes or the like. Administrationcan also occur through an inhalant, such as by nasal route or aerosol,which can be important when a subject is suffering from cystic fibrosis.Also preferred, the dosage is repeatedly administered. Still preferred,the MN3 antibody is administered in a dosage of 20 to 2000 milligramsprotein per dose. Even more preferred, the MN3 antibody is administeredin a dosage of 50 to 500 mg protein dose per injection.

The compositions for treatment contain at least one naked murine,humanized, chimeric or human MN3 antibody described herein or fragmentthereof alone or in combination with other MN3 antibodies or antibodyfragments thereof, such as other MN3 humanized, chimeric or humanantibodies. Preferably, the MN3 antibody, fusion protein, or fragmentthereof in the composition for treatment is administered in a dosage of20-2000 milligrams per dose. Also preferred, the MN3 antibody orfragment thereof in the composition for treatment is an MN3 antibody orfragment thereof. The present invention also contemplates treatment withat least one naked humanized, chimeric or human MN3 antibody or fragmentthereof in combination with other antibodies or antibody fragmentsthereof that are not MN3 antibodies, whereby these other antibodies canbe administered unconjugated (naked) or conjugated with at least onediagnostic/detection or therapeutic agent. For example, other antibodiessuitable for combination therapy include, but are not limited to,granulocyte associated antibodies and fragments thereof such asantibodies reactive with MAbs reactive with NCA-90, NCA-95, CD15, CD33,and from the MAbs MN2, MN3, MN-15, NP-1, NP-2, BW 250/183, and MAb 47,or a combination thereof. Suitable antibodies could also include thosetargeted against myeloid leukemias. Additionally, treatment can beeffected with at least one humanized, chimeric or human MN3immunoconjugate or fragment thereof alone or in combination withanti-granulocyte antibodies or antibody fragments thereof. Stillpreferred, compositions for treatment can contain at least onehumanized, chimeric or human MN3 immunoconjugate or fragment thereof incombination with other antibodies or antibody fragments thereof that arenot anti-granulocyte antibodies, these being either naked or conjugatedto a therapeutic agent.

Similarly, conjugated and naked MN3 humanized, chimeric or humanantibodies or fragments thereof may be used alone or may be administeredwith, but unconjugated to, the various diagnostic/detection ortherapeutic agents described herein. Also, naked or conjugated MN3antibodies to the same or different epitope or antigen may be alsocombined with one or more of the antibodies of the present invention.

Accordingly, the present invention contemplates the administration ofmurine, humanized, chimeric and human MN3 antibodies and fragmentsthereof alone, as a naked antibody, or administered as a multimodaltherapy. Multimodal therapies of the present invention further includeimmunotherapy with naked or conjugated MN3 antibodies supplemented withadministration of other conjugated or unconjugated antibody, fusionprotein, or fragment thereof. For example, a humanized, chimeric orhuman MN3 antibody may be combined with another naked humanized, nakedchimeric or naked human MN3 antibody, or a humanized, chimeric or humanMN3 antibody immunoconjugate, such as a humanized, chimeric or human MN3antibody conjugated to an isotope, one or more chemotherapeutic agents,cytokines, enzymes, enzyme-inhibitors, hormones or hormone antagonists,metals, toxins, or a combination thereof. For example, the presentinvention contemplates treatment of a naked or conjugated MN3 antibodyor fragment thereof before, in combination with, or after otheranti-granulocyte associated antibodies. A fusion protein of a murine,humanized, chimeric or human MN3 antibody and a toxin or may also beused in this invention. Many different antibody combinations may beconstructed, either as naked antibodies or as partly naked and partlyconjugated with a therapeutic agent or immunomodulator, or merely incombination with another therapeutic agents, such as a cytotoxic drug orwith radiation.

The compositions for treatment contain at least one humanized, chimericor human monoclonal MN3 antibody or fragment thereof alone or incombination with other antibodies and fragments thereof, such as othernaked or conjugated, murine, humanized, chimeric, or human antibodies,or fragments thereof, or fusion proteins or fragments thereof, ortherapeutic agents. In particular, combination therapy with a fullyhuman antibody is also contemplated and is produced by the methods asset forth above.

Naked or conjugated antibodies, fusion proteins, or fragments thereofmay be also combined with one or more of the antibodies, fusionproteins, or fragments thereof to the same or different epitope orantigen. For example, a naked, murine, humanized, chimeric or human MN3antibody may be combined with a naked murine, humanized, naked chimericor naked human MN3 antibody; a murine, humanized, chimeric or humannaked MN3 antibody may be combined with a MN3 immunoconjugate; a nakedmurine, humanized, chimeric, human MN3 antibody may be combined with adifferent antibody radioconjugate or a different naked antibody; amurine, humanized, chimeric or fully human MN3 antibody may be combinedwith a murine, humanized, chimeric or human MN3 antibody conjugated toan isotope, or to one or more chemotherapeutic agents, cytokines orother immunomodulator, toxins, enzymes, enzyme inhibitors, hormones,hormone antagonists, or a combination thereof. A fusion protein of amurine, humanized, chimeric or human MN3 antibody and a toxin orimmunomodulator may also be used in this invention. Many differentantibody combinations, targeting at least two different antigens may beconstructed, either as naked antibodies or as partly naked and partlyconjugated with a therapeutic agent or immunomodulator, or merely incombination with another therapeutic agents, such as a cytotoxic drug orwith radiation.

Multimodal therapies of the present invention further includeimmunotherapy with naked MN3 antibodies or fragments thereofsupplemented with administration of granulocyte associated antibodies inthe form of a conjugated or unconjugated antibody, fusion proteins, orfragment thereof. In a preferred embodiment, antibodies or fragmentsthereof for multimodal therapy include, but are not limited to,antibodies reactive with NCA-90, NCA-95, CD15, CD33, and from the MAbsMN2, MN3, MN-15, NP-1, NP-2, BW 250/183, and MAb 47, or a combinationthereof. These antibodies include polyclonal, monoclonal, chimeric,human or humanized antibodies and fragments thereof that recognize atleast one epitope on these antigenic determinants.

In another form of multimodal therapy, subjects receive naked MN3antibodies or fragments thereof, and/or MN3 immunoconjugates orfragments thereof, in conjunction with standard cancer chemotherapy.Fludarabine, alone or in combination with cytosine arabinoside, is aregimen used to treat myeloid leukemia. Other suitable combinationchemotherapeutic regimens are well known, such as with chlorambucilalone, or in combination with these other drugs, to those of skill inthe art. In a preferred multimodal therapy, both chemotherapeutic drugsand cytokines are co-administered with a conjugated or unconjugated MN3antibody, fusion protein, or fragment thereof, according to the presentinvention. Preferably, the MN3 antibody or fragment thereof is an MN3antibody or fragment thereof. The cytokines, chemotherapeutic drugs andantibody, fusion protein, or fragment thereof, can be administered inany order, or together.

The present invention also encompasses the use of the bsAb and at leastone therapeutic or diagnostic/detection agent associated with the linkermoieties discussed above in intraoperative, intravascular, andendoscopic tumor and lesion detection, biopsy and therapy as describedin U.S. Pat. No. 6,096,289, and incorporated herein by reference.Preferably, the bispecific antibody has at least one arm that binds theepitope recognized by MN3.

The MN3 antibodies, fusion proteins, and fragments thereof of thepresent invention can be employed not only for therapeutic or imagingpurposes, but also as aids in performing research in vitro. For example,the bsAbs of the present invention can be used in vitro to ascertain ifa targetable construct can form a stable complex with one or more bsAbs.Such an assay would aid the skilled artisan in identifying targetableconstructs which form stable complexes with bsAbs. This would, in turn,allow the skilled artisan to identify targetable constructs which arelikely to be superior as therapeutic and/or imaging agents.

The assay is advantageously performed by combining the targetableconstruct in question with at least two molar equivalents of a bsAb.Following incubation, the mixture is analyzed by size-exclusion HPLC todetermine whether or not the construct has bound to the bsAb.Alternatively, the assay is performed using standard combinatorialmethods wherein solutions of various bsAbs are deposited in a standard96-well plate. To each well, is added solutions of targetableconstruct(s). Following incubation and analysis, one can readilydetermine which construct(s) bind(s) best to which bsAb(s).

It should be understood that the order of addition of the bsAb to thetargetable construct is not crucial; that is, the bsAb may be added tothe construct and vice versa. Likewise, neither the bsAb nor theconstruct needs to be in solution; that is, they may be added either insolution or neat, whichever is most convenient. Lastly, the method ofanalysis for binding is not crucial as long as binding is established.Thus, one may analyze for binding using standard analytical methodsincluding, but not limited to, FABMS, high-field NMR or otherappropriate method in conjunction with, or in place of, size-exclusionHPLC.

Bispecific Antibody Therapy and Diagnosis

The present invention provides a bispecific antibody or antibodyfragment having at least one binding region that specifically binds atargeted cell marker and at least one other binding region thatspecifically binds a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment.

For example, a method of treating or identifying diseased tissues in asubject, comprising: (A) administering to said subject a bi-specificantibody or antibody fragment having at least one arm that specificallybinds a targeted tissue and at least one other arm that specificallybinds a targetable conjugate, wherein said one arm that specificallybinds a targeted tissue is an MN3 antibody; (B) optionally,administering to said subject a clearing composition, and allowing saidcomposition to clear non-localized antibodies or antibody fragments fromcirculation; (C) administering to said subject a first targetableconjugate which comprises a carrier portion which comprises or bears atleast one epitope recognizable by said at least one other arm of saidbi-specific antibody or antibody fragment, and one or more conjugatedtherapeutic or diagnostic agents; and (D) when said therapeutic agent isan enzyme, further administering to said subject 1) a prodrug, when saidenzyme is capable of converting said prodrug to a drug at the targetsite; or 2) a drug which is capable of being detoxified in said subjectto form an intermediate of lower toxicity, when said enzyme is capableof reconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 3) a prodrug which is activated in said subject through naturalprocesses and is subject to detoxification by conversion to anintermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 4) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by said atleast one other arm of said bi-specific antibody or antibody fragment,and a prodrug, when said enzyme is capable of converting said prodrug toa drug at the target site, is described. Optionally, when said firsttargetable conjugate comprises a prodrug, administering a secondtargetable conjugate which comprises a carrier portion which comprisesor bears at least one epitope recognizable by said at least one otherarm of said bi-specific antibody or antibody or antibody fragment, andan enzyme capable of converting said prodrug to a drug or ofreconverting a detoxified intermediate of said drug to a toxic form.Preferably, the targetable conjugate comprises at least two HSG haptens.

In a related vein, a method for detecting or treating neoplasmsexpressing an antigen recognized by MN3 in a mammal is described. Thismethod comprises (A) administering an effective amount of a bispecificantibody or antibody fragment comprising at least one arm thatspecifically binds a targeted tissue and at least one other arm thatspecifically binds a targetable conjugate, wherein said one arm thatspecifically binds a targeted tissue is an MN3 antibody or fragmentthereof; and (B) administering a targetable conjugate selected from thegroup consisting of (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG) -NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (SEQ ID NO: 7); (iii)Ac-Lys(HSG)D-Tyr -Lys(HSG)-Lys(Tscg-Cys)-NH₂;

Optionally, the method further comprises administering to a subject aclearing composition, and allowing the composition to clearnon-localized antibodies or antibody fragments from the circulation.

Bispecific antibodies and fragments thereof of the present invention areuseful in pretargeting methods and provide a preferred way to delivertwo therapeutic agents or two diagnostic/detection agents to a subject.U.S. Ser. No. 09/382,186 discloses a method of pretargeting using abispecific antibody, in which the bispecific antibody is labeled with¹²⁵I and delivered to a subject, followed by a divalent peptide labeledwith ^(99m)Tc. The delivery results in excellent tumor/normal tissueratios for ¹³¹I and ^(99m)Tc, thus showing the utility of two diagnosticradioisotopes. Any combination of known therapeutic agents or diagnosticagents can be used to label the MN3 antibodies, MN3 fusion proteins, andfragments thereof of the present invention. The binding specificity ofthe MN3 immunoconjugate, the efficacy of the therapeutic agent ordiagnostic agent and the effector activity of the Fc portion of theantibody can be determined by standard testing of the conjugates.

The administration of a bsAb and a therapeutic agent associated with thelinker moieties discussed above may be conducted by administering thebsAb at some time prior to administration of the therapeutic agent whichis associated with the linker moiety. The doses and timing of thereagents can be readily devised by a skilled artisan, and are dependenton the specific nature of the reagents employed. If a bsAb-F(ab′)₂derivative is given first, then a waiting time of 24-72 hr beforeadministration of the linker moiety would be appropriate. If an IgG-Fab′bsAb conjugate is the primary targeting vector, then a longer waitingperiod before administration of the linker moiety would be indicated, inthe range of 3-10 days.

After sufficient time has passed for the bsAb to target to the diseasedtissue, the diagnostic/detection agent is administered. Subsequent toadministration of the diagnostic/detection agent, imaging can beperformed. Tumors can be detected in body cavities by means of directlyor indirectly viewing various structures to which energy of theappropriate wavelength is delivered and then collected. Lesions at anybody site can be viewed so long as nonionizing radiation or energy canbe delivered and recaptured from these structures. For example, PET,which is a high resolution, non-invasive, imaging technique, can be usedwith the inventive antibodies for the visualization of human disease. InPET, 511 keV gamma photons produced during positron annihilation decayare detected.

The linker moiety may also be conjugated to an enzyme capable ofactivating a prodrug at the target site or improving the efficacy of anormal therapeutic by controlling the body's detoxification pathways.Following administration of the bsAb, an enzyme conjugated to the linkermoiety, a low MW hapten recognized by the second arm of the bsAb, isadministered. After the enzyme is pretargeted to the target site, acytotoxic drug is injected, which is known to act at the target site.The drug may be one which is detoxified by the mammal's ordinarydetoxification processes. For example, the drug may be converted intothe potentially less toxic glucuronide in the liver. The detoxifiedintermediate can then be reconverted to its more toxic form by thepretargeted enzyme at the target site. Alternatively, an administeredprodrug can be converted to an active drug by the pretargeted enzyme.The pretargeted enzyme improves the efficacy of the treatment byrecycling the detoxified drug. This approach can be adopted for use withany enzyme-drug pair.

The enzyme capable of activating a prodrug at the target site orimproving the efficacy of a normal therapeutic by controlling the body'sdetoxification pathways may alternatively be conjugated to the hapten.The enzyme-hapten conjugate is administered to the subject followingadministration of the pre-targeting bsAb and is directed to the targetsite. After the enzyme is localized at the target site, a cytotoxic drugis injected, which is known to act at the target site, or a prodrug formthereof which is converted to the drug in situ by the pretargetedenzyme. As discussed above, the drug is one which is detoxified to forman intermediate of lower toxicity, most commonly a glucuronide, usingthe mammal's ordinary detoxification processes. The detoxifiedintermediate, e.g., the glucuronide, is reconverted to its more toxicform by the pretargeted enzyme and thus has enhanced cytotoxicity at thetarget site. This results in a recycling of the drug. Similarly, anadministered prodrug can be converted to an active drug through normalbiological processes. The pretargeted enzyme improves the efficacy ofthe treatment by recycling the detoxified drug. This approach can beadopted for use with any enzyme-drug pair.

The invention further contemplates the use of the inventive bsAb and thediagnostic agent(s) in the context of Boron Neutron Capture Therapy(BNCT) protocols. BNCT is a binary system designed to deliver ionizingradiation to tumor cells by neutron irradiation of tumor-localized ¹⁰Batoms. BNCT is based on the nuclear reaction which occurs when a stableisotope, isotopically enriched ¹⁰B (present in 19.8% natural abundance),is irradiated with thermal neutrons to produce an alpha particle and a⁷Li nucleus. These particles have a path length of about one celldiameter, resulting in high linear energy transfer. Just a few of theshort-range 1.7 MeV alpha particles produced in this nuclear reactionare sufficient to target the cell nucleus and destroy it. Success withBNCT of cancer requires methods for localizing a high concentration of¹⁰B at tumor sites, while leaving non-target organs essentiallyboron-free. Compositions and methods for treating tumors in subjectsusing pre-targeting bsAb for BNCT are described in co-pending patentapplication Ser. No. 09/205,243, incorporated herein in its entirety andcan easily be modified for the purposes of the present invention.

A clearing agent may be used which is given between doses of the bsAband the linker moiety. The present inventors have discovered that aclearing agent of novel mechanistic action may be used with theinvention, namely a glycosylated anti-idiotypic (anti-Id) Fab′ fragmenttargeted against the disease targeting amm(s) of the bsAb. For example,anti-CSAp (Mu-9 Ab)×anti-peptide bsAb is given and allowed to accrete indisease targets to its maximum extent. To clear residual bsAb, ananti-idiotypic (anti-Id) Ab to Mu-9 is given, preferably as aglycosylated Fab′ fragment. The clearing agent binds to the bsAb in amonovalent manner, while its appended glycosyl residues direct theentire complex to the liver, where rapid metabolism takes place. Thenthe therapeutic which is associated with the linker moiety is given tothe subject. The anti-Id Ab to the Mu-9 arm of the bsAb has a highaffinity and the clearance mechanism differs from other disclosedmechanisms (see Goodwin et al., ibid), as it does not involvecross-linking, because the anti-Id-Fab′ is a monovalent moiety. The sameis accomplished with MN3 Mabs and corresponding anti-MN3 anti-Idantibodies.

Also contemplated herein is a kit useful for treating or identifyingdiseased tissues in a subject comprising: (A) a bi-specific antibody orantibody fragment having at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds atargetable conjugate, wherein said one arm that specifically binds atargeted tissue is an MN3 antibody or fragment thereof; (B) a firsttargetable conjugate which comprises a carrier portion which comprisesor bears at least one epitope recognizable by said at least one otherarm of said bi-specific antibody or antibody fragment, and one or moreconjugated therapeutic or diagnostic agents; and (C) optionally, aclearing composition useful for clearing non-localized antibodies andantibody fragments; and (D) optionally, when said therapeutic agentconjugated to said first targetable conjugate is an enzyme, 1) aprodrug, when said enzyme is capable of converting said prodrug to adrug at the target site; or 2) a drug which is capable of beingdetoxified in said subject to form an intermediate of lower toxicity,when said enzyme is capable of reconverting said detoxified intermediateto a toxic form, and, therefore, of increasing the toxicity of said drugat the target site, or 3) a prodrug which is activated in said subjectthrough natural processes and is subject to detoxification by conversionto an intermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 4) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by said atleast one other arm of said bi-specific antibody or antibody fragment,and a prodrug, when said enzyme is capable of converting said prodrug toa drug at the target site. Preferably, the targetable conjugate isselected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (SEQ ID NO: 7); (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

A method of screening for a targetable conjugate is also described,comprising (A) contacting said targetable construct with a bi-specificantibody or antibody fragment having at least one arm that specificallybinds a targeted tissue and at least one other arm that specificallybinds said targetable conjugate to give a mixture, wherein said one armthat specifically binds a targeted tissue is a MN3 antibody or fragmentthereof; and (B) optionally incubating said mixture; and (C) analyzingsaid mixture.

The present invention further provides a method for imaging malignanttissue or cells in a mammal expressing an antigen recognized by MN3; amethod of intraoperatively identifying/disclosing diseased tissuesexpressing an antigen recognized by MN3, in a subject; a method forendoscopic identification of diseased tissues expressing an antigenrecognized by MN3, in a subject and a method for the intravascularidentification of diseased tissues expressing an antigen recognized byMN3, in a subject. Such methods comprise (A) administering an effectiveamount of a bispecific antibody or antibody fragment comprising at leastone arm that specifically binds a targeted tissue expressing an antigenrecognized by MN3 and at least one other arm that specifically binds atargetable conjugate, wherein said one arm that specifically binds atargeted tissue is an MN3 antibody or fragment thereof; and (B)administering a targetable conjugate selected from the group consistingof (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ (SEQ ID NO: 7); (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

Also considered herein is a method of detection of lesions during anendoscopic, laparoscopic, intravascular catheter, or surgical procedure,wherein the method comprises: (A) injecting a subject who is to undergosuch a procedure with a bispecific antibody F(ab)₂ or F(ab′)₂ fragment,wherein the bispecific antibody or fragment has a first antibody bindingsite which specifically binds to an antigen recognized by MN3, and has asecond antibody binding site which specifically binds to a hapten, andpermitting the antibody fragment to accrete at target sites; (B)optionally clearing non-targeted antibody fragments using agalactosylated anti-idiotype clearing agent if the bispecific fragmentis not largely cleared from circulation within about 24 hours ofinjection, and injecting a bivalent labeled hapten, which quicklylocalizes at the target site and clears through the kidneys; (C)detecting the presence of the hapten by close-range detection ofelevated levels of accreted label at the target sites with detectionmeans, within 48 hours of the first injection, and conducting saidprocedure, wherein said detection is performed without the use of acontrast agent or subtraction agent. Preferably, the hapten is labeledwith a diagnostic/detection radioisotope, a MRI image-enhancing agent ora fluorescent label.

In a related vein, a method for close-range lesion detection, during anoperative, intravascular, laparoscopic, or endoscopic procedure, whereinthe method comprises: (A) injecting a subject to such a procedureparenterally with an effective amount of an MN3 immunoconjugate orfragment thereof, (B) conducting the procedure within 48 hours of theinjection; (C) scanning the accessed interior of the subject at closerange with a detection means for detecting the presence of said labeledantibody or fragment thereof; and (D) locating the sites of accretion ofsaid labeled antibody or fragment thereof by detecting elevated levelsof said labeled antibody or fragment thereof at such sites with thedetection means, is also described.

9. Pharmaceutically Suitable Excipients

The murine, humanized, chimeric and human MN3 MAbs to be delivered to asubject can consist of the MAb alone, immunoconjugate, fusion protein,or can comprise one or more pharmaceutically suitable excipients, one ormore additional ingredients, or some combination of these.

The conjugated or unconjugated MN3 antibodies and fragments thereof, orfusion proteins and fragments thereof, of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions. Preferably, the MN3 antibody or fragment thereof is an MN3antibody or fragment thereof. Sterile phosphate-buffered saline is oneexample of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The conjugated or unconjugated MN3 antibody, fusion protein, orfragments thereof of the present invention can be formulated forintravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the MN3 antibody or fragments is an MN3antibody or fragment thereof. Formulations for injection can bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic/detectionimmunoconjugate or naked antibody, fusion protein, or fragments thereof.Control release preparations can be prepared through the use of polymersto complex or adsorb the immunoconjugate or naked antibody. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992).The rate of release of an immunoconjugate or antibody from such a matrixdepends upon the molecular weight of the immunoconjugate or antibody,the amount of immunoconjugate, antibody within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. J 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The conjugated or unconjugated MN3 antibody, fusion protein, orfragments thereof may also be administered to a mammal subcutaneously oreven by other parenteral routes. Moreover, the administration may be bycontinuous infusion or by single or multiple boluses. In general, thedosage of an administered immunoconjugate, or naked antibody, fusionprotein or fragments thereof for humans will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Typically, it is desirable toprovide the recipient with a dosage of immunoconjugate, naked antibodyfusion protein, naked antibody, or fragments thereof that is in therange of from about 0.3 mg/kg to 30 mg/kg as a single intravenousinfusion, although a lower or higher dosage also may be administered ascircumstances dictate. This dosage may be repeated as needed, forexample, once per week for 4-10 weeks, preferably once per week for 8weeks, and more preferably, once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months. Thedosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

For purposes of therapy, the conjugated or unconjugated antibody, fusionprotein, or fragment thereof is administered to a mammal in atherapeutically effective amount. Preferably, the MN3 antibody orfragment thereof is an MN3 antibody or fragment thereof. A suitablesubject for the present invention is usually a human, although anon-human animal subject is also contemplated. An antibody preparationis said to be administered in a “therapeutically effective amount” ifthe amount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient mammal. In particular, anantibody preparation of the present invention is physiologicallysignificant if its presence invokes an antitumor response or mitigatesthe signs and symptoms of an autoimmune disease state. A physiologicallysignificant effect could also be the evocation of a humoral and/orcellular immune response in the recipient mammal.

10. Expression Vectors

The DNA sequence encoding a murine, humanized, chimeric or human MN3 MAbcan be recombinantly engineered into a variety of known host vectorsthat provide for replication of the nucleic acid. These vectors can bedesigned, using known methods, to contain the elements necessary fordirecting transcription, translation, or both, of the nucleic acid in acell to which it is delivered. Known methodology can be used to generateexpression constructs the have a protein-coding sequence operably linkedwith appropriate transcriptional/translational control signals. Thesemethods include in vitro recombinant DNA techniques and synthetictechniques. For example, see Sambrook et al., 1989, MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory (New York); Ausubel etal., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons(New York). Also provided for in this invention is the delivery of apolynucleotide not associated with a vector.

Vectors suitable for use in the instant invention can be viral ornon-viral. Particular examples of viral vectors include adenovirus, AAV,herpes simplex virus, lentivirus, and retrovirus vectors. An example ofa non-viral vector is a plasmid. In a preferred embodiment, the vectoris a plasmid.

An expression vector, as described herein, is a polynucleotidecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements.

Preferably, the expression vector of the instant invention comprises theDNA sequence encoding a humanized, chimeric or human MN3 MAb, whichincludes both the heavy and the light chain variable and constantregions. However, two expression vectors may be used, with onecomprising the heavy chain variable and constant regions and the othercomprising the light chain variable and constant regions. Stillpreferred, the expression vector further comprises a promoter, a DNAsequence encoding a secretion signal peptide, a genomic sequenceencoding a human Ig light or heavy chain constant region, an Ig enhancerelement and at least one DNA sequence encoding a selection marker.

The following applications, which describe methods in which the presentantibodies and fragments can be used and alternative embodiments for thepresent antibodies and fragments, are also incorporated herein byreference: 60/328,835; 60/341,881; 60/342,103; 60/345,641; 60/404,919;60/436,359; 60/464,532; U.S. Ser. Nos. 10/270,071; 10/270,073;10/328,190; PCT/U502/32717; PCT/US02/32718; and PCT/US02/38985.

The invention is further described by reference to the followingexamples, which are provided for illustration only. The invention is notlimited to the examples but rather includes all variations that areevident from the teachings provided herein. All citations made topublished articles, as well as to patents and patent applications, areincorporated herein in their entirety.

EXAMPLES Example 1 Molecular Cloning and Sequence Elucidation for MN3Heavy and Light Chain Variable Regions

The VH and Vκ genes of MN3 were obtained by RT-PCR as described byOrlandi et al. (PNAS 86:3833-3837 (1989) and Leung et al. (Hybridoma13:469-476 (1994). Multiple independent clones were sequenced toeliminate possible errors resulted from PCR reaction. The codingsequences for murine VH and Vκ were found in the cloned PCR products anddesignated as MN3Vκ (FIG. 1A) and MN3VH (FIG. 1B), respectively. Toconfirm the authenticity of V genes for MN3, a chimeric MN3 antibody wasconstructed and expressed in Sp2/0 cell. The cloned Vκ and VH fragmentswere first subcloned into the respective staging vectors, VKpBR andVHpBS (FIGS. 2A and 2B). The 650 bp fragment containing the Vκ, alongwith the promoter and signal peptide sequences, were then excised fromthe VKpBR staging vector by HindIII and BamHI restriction endonucleasesdigestion and subcloned into pKh, which is a pSVhyg-based expressionvector containing the genomic sequence of the human kappa constantregion, an Ig enhancer, a kappa enhancer and the hyg-resistant gene,resulting in the final expression vector for the light chain of cMN3.Similarly, the about 850 bp VH fragments was subcloned into pG1g, apSVgpt-based expression vector carrying the genomic sequence of thehuman IgG1 constant region, an Ig enhancer and the xanthine-guaninephosphoribosyl transferase (gpt) gene, resulting in the final expressionvector for the heavy chain of cMN3. The two expression vectors wereco-transfected into Sp2/0-Ag14 cells by electroporation and selected forhygromycin resistance. Stable transfectoma clones were expanded andconfirmed to produce chimeric antibodies by ELISA assay (Example 3). Theantigen binding specificity and affinity of purified cMN3 were evaluatedby a competitive binding ELISA assay. Briefly, varying concentrations ofcMN3 or mMN3 were mixed with a constant amount of biotinylated murineMN3 and incubated in 96-well ELISA plate coated with CEA. The residualbinding of the biotinylated MN3 was measured by HRP-conjugatedstreptavidin and a substrate solution containing ortho-phenylenediaminedihydrochloride and H₂O₂. As shown in FIG. 3, cMN3 competed with withradiolabeled HRP-conjugated MN3 for antigen binding and the bindingactivity of cMN3 is comparable with that of MN3.

Example 2 Choice of Human Frameworks and Sequence Design for hMN3

The light and heavy chain variable region sequences encoding thehumanized MN3 antibody (hMN3) were designed and constructed. Bycomparing the murine MN3 V region FR sequences to that of human Abs inthe Kabat database (Sequences of Proteins of Immunological Interest(Bethesda, Md.: U.S. Department of Health and Human Services, PublicHealth Service, National Institute of Health, 1991), the FRs of humanREI and EU VH were found to exhibit the highest degree of sequencehomology to that of MN3Vκ and MN3VH, respectively (FIG. 4). Oneexception is the FR4 of MN3VH, which showed the highest sequencehomology with that of KOL VH (FIG. 4B). Thus, the human REI frameworksequences was used for Vκ, and a combination of EU (FR1-3) and KOL (FR4)for VH as shown in FIGS. 4A and 4B, respectively. A variable number ofmurine amino acid residues in each chain outside of the CDR regions weremaintained in the humanized design when compared to the starting humanantibody frameworks. The light chain of hMN3 contains six amino acidchanges from the REI framework: T20S, T22S, T39K, S60D, Y71F, and Q100G(FIG. 4A). The heavy chain of hMN3 also contains six changes from thehuman EU frameworks: G27Y, S30R, V67F, T68A, I69F, and G94R, (FIG. 4B).

Example 3 Method of hMN3 Construction

Each humanized variable chain was constructed in two parts, a 5′- and a3′-half, designated as “A” and “B”, respectively. Each half was producedby PCR amplification of a single stranded long synthetic oligonucleotidetemplate with two short flanking primers using Taq polymerase. Theamplified fragments were first cloned into the pCR4 TA cloning vectorfrom Invitrogen and subjected to DNA sequencing. The templates andprimer pairs are listed as follows:

Template Primers Product Olgo G Oligo 13/Oligo 14 VHA Oligo H Oligo15/Oligo 16 VHB Oligo I Oligo 17/Oligo 18 VKA Oligo J Oligo 19/Oligo 20VKB

The sequence information for the above identified oligonucleotides is asfollows:

Oligo G (represents the minus strand of hMN3VH domain complementary tont 25-173, 149 bp) (SEQ ID NO: 38) 5′-GGCTCACCGG TGTAGGTGTT TATCCAGCCCATCCACTCTA AACCCTGTCC TGGAGCCTGT CTCACCCAGT TCATTCCATA GTTTCTGAAGGTATACCCAG AAGCCTTGCA GGAGACCTTG ACGCTAGATC CAGGCTTCTT GACCTCAGC-3′Oligo H (represents the minus strand of hMN3VH domain complementary tont 181-329, 149 bp) (SEQ ID NO: 39) 5′-TCGAGGCTAC TACCGTTGAA ATCCATCCATCCCTTTCTTG CACAGAAATA GAAAGCCGTG TCCTCAGATC TCAAGCTAGA CAGCTCCATATAGGCAGTGT TGGTAGATTC GTCGGCTGTG AAGGCAAACC GTCCCTTGAA GTCATCAGC-3′Oligo 13 (SEQ ID NO: 40) 5′-CCAACTGCAG CAGTCTGGAG CTGAGGTCAA GAAGCCT-3′Oligo 14 (SEQ ID NO: 41) 5′-GGCTCACCGG TGTAGGTGTT-3′ Oligo 15 (SEQ IDNO: 42) 5′-ACCTACACCG GTGAGCCAAC ATATGCTGAT GACTTCAAGG GACG-3′ Oligo 16(SEQ ID NO: 43) 5′-GGTGACCGGG GTCCCTTGGC CCCAGTAGTC GAGGCTACTACCGTTGA-3′ Oligo I (represents the minus strand of hMN3Vk domaincomplementary to nt 31-170, 140 bp) (SEQ ID NO: 44) 5′-GAAACTTTGTAGATCAGCAG CTTTGGAGCC TTACCTGGCT TCTGCTGGTA CCATTCTAAA TAGGTGTTTCCATTACTATG TACAATGCTC TGACTGGATC TACAAGAGAT GGACACTCTG TCACCCACGCTGGCGCTCAG-3′ Oligo J (represents the minus strand of hMN3VH domaincomplementary to nt 191-321, 131 bp) (SEQ ID NO: 45) 5′-GGTCCCGCCGCCGAACGTCG GAGGAACATG TGAACCTTGA AAGCAGTAGT AGGTGGCGAT GTCCTCTGGCTGGAGGCTGC TGATGGTGAA GGTGAAGTCG GTACCGCTAC CGCTACCGCT GAATCTGTCT G-3′Oligo 17 (SEQ ID NO: 46) 5′-CAGCTGACCC AGAGCCCAAG CAGCCTGAGC GCCAGCGTGGG-3′ Oligo 18 (SEQ ID NO: 47) 5′-CTGGCACTCC GGAAAATCGG TTGGAAACTTTGTAGATCAG CAG-3′ Oligo 19 (SEQ ID NO: 48) 5′-CAACCGATTT TCCGGAGTGCCAGACAGATT CAGCGGT-3′ Oligo 20 (SEQ ID NO: 49) 5′-GATCTCCACC TTGGTCCCGCCGCCGAACGT CGG-3′1. Light Chain

Unique restriction sites were included at the ends of each fragment tofacilitate joining through DNA ligation. The amplified VKA fragmentcontained a PvuII restriction site, CAGCTG, at its 5′-end and a BspEIrestriction site, TCCGGA, at the 3′-end. The amplified VKB fragmentcontained a BspEI restriction site at its 5′-end and a BglII restrictionsite, AGATCT, at the 3′-end. Assembly of the full-length Vκ chain wasaccomplished by restriction enzyme digestion of each fragment with theappropriate 5′- and 3′-enzymes and ligation into the VKpBR2 vectorpreviously digested with PvuII and BclI (BclI digested end is compatiblewith that of BglII). The resulting ligated product contains the Afragment ligated to the PvuII site, the B fragment ligated to the BclIsite, and the A and B fragments joined together at the BstBI site (FIG.5A). Upon confirmation of a correct open reading frame by DNAsequencing, the intact chain was removed from VKpBR2 as a XbaI to BamHIfragment and ligated into the pdHL2 expression vector. The vectorcontaining only Vκ sequence was designated as hMN3VkpdHL2.

2. Heavy Chain

The same construction method as done for Vκ was carried out VH with thefollowing modifications. The 5′-end restriction site of the A fragmentswas Pstl (CTGCAG) and the 3′-end restriction site of B fragments wasBstEll (GGTCACC). These fragments were joined together upon ligationinto the VHpBS2 vector at a common Agel site (ACCGGT), resulting infull-length VH sequences (FIG. 5B), which were confirmed by DNAsequencing. The assembled VH genes were subcloned as XhoI-BamHIrestriction fragments into the expression vector containing the VKsequence, hMN3VkpdHL2, predigested with XhoI and HindIII. To ligate theBamHI end of the VH fragment to the HindIII end of the vector, a linker,designated as HNB was used. The resulting expression vectors weredesignated as hMN3pdHL2.

HNB linker 5′-AGCTTGCGGCCGC-3′ (SEQ ID NO: 50) 3′-ACGCCGGCGCTAG-5′ (SEQID NO: 51)

Example 4 Transfection and Expression of hMN3 Antibodies

The procedure employed to express hMN3 using pdHL2 based vecto in Sp2/0cells by transfection was the same as described by Qu et al. Clin.Cancer Res. 5:3095s-3100s (1999). Briefly, 30 ug of hMN3pdHL2 werelinearized by digestion with SalI and transfected into Sp2/0-Ag14 cellsby electroporation (450V and 25 uF). The transfected cells were platedinto 96-well plate for 2 days and then selected for MTX resistance.Supernatants from colonies surviving selection were monitored for humanantibody secretion by ELISA assay. Positive cell clones were expandedand hMN3 was purified from cell culture supernatant by affinitychromatography on a Protein A column.

Example 5 Binding Activity Assays

A competition ELISA binding assay was carried out to assess theimmunoreactivity of hMN3 relative to the parent MN3, and cMN3. 96-wellmicrotitration plate was coated with CEA. Varying concentrations of MN3,cMN3 or hMN3 (0.01-100 ug/ml) was made to compete with a constant amountof biotinylated murine MN3 (0.5 ug/ml) for binding to CEA, and theresidual binding of the biotinylated MN3 in the presence of thecompeting antibodies was measured. As shown in FIG. 6, hMN3 exhibitedcomparable binding activity as that of murine MN3 and cMN3.

Example 6 Therapy of Acute Myeloid Leukemia

A 71-year-old man with a history of acute myeloid leukemia relapsesfollowing 2 courses of chemotherapy, and presents with a high number ofimmature, granulocytic leukemia cells in his blood and marrow, and alsowith an enlarged spleen, anemia, malaise, lethargy, diffuse bone pain,and some increased bruising and bleeding. He is first given a 100 mCidose of I-131 conjugated to the humanized MN3 antibody (50 mg ofantibody given as part of the dose). Peripheral blood counts 4 weekslater indicate a fall in myeloid leukemic cells by 60% and animprovement in his splenomegaly. He is then given, 2 weeks later, 4weekly doses of naked, humanized MN3 antibody in 3-hr i.v. infusions,each dose being 450 mg of the antibody. This cycle of 4 doses is thenrepeated 3 months later. His myeloid leukemia cells in the blood are notreduced by another 30% from the last measurement, and a bone marrowbiopsy shows, in contrast to the pre-treatment biopsy, a markedreduction in infiltration with leukemic cells. The patient's other signsand symptoms improve over the next 2 months.

The present methods can involve any or all of the steps or conditionsdiscussed above in various combinations, as desired. Accordingly, itwill be readily apparent to the skilled artisan that in some of thedisclosed methods certain steps can be deleted or additional stepsperformed without affecting the viability of the methods.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Accordingly, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention.

All references disclosed herein are specifically incorporated byreference thereto. Unless otherwise specified, “a” or “an” means “one ormore”. While preferred embodiments have been illustrated and described,it should be understood that changes and modifications can be madetherein in accordance with ordinary skill in the art without departingfrom the invention in its broader aspects as defined in the claims.

1. A chimeric or humanized MN-3 antibody or fragment thereof that bindsNCA90 comprising the MN-3 light chain CDR sequences CDR1(RSSQSIVHSNGNTYLE, SEQ ID NO:1), CDR2 (KVSNRFS, SEQ ID NO:2) and CDR3(FQGSHVPPT, SEQ ID NO:3) and the MN-3 heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:4), CDR2 (WINTYTGEPTYADDFKG, SEQ ID NO:5) and CDR3(KGWMDFNSSLDY, SEQ ID NO:6).
 2. The humanized antibody or fragmentthereof of claim 1, wherein the antibody or fragment comprises theframework (FR) region sequences of the light and heavy chain variableregions of a human antibody and at least one light and heavy chainconstant regions of a human antibody.
 3. The humanized antibody orfragment thereof of claim 2, wherein at least one of the FRs of thelight and heavy chain variable regions of the humanized MN-3 antibody orfragment thereof comprises at least one amino acid substituted with thecorresponding amino acid of the murine MN-3 antibody.
 4. The humanizedantibody or fragment thereof of claim 3, wherein the at least one aminoacid from the murine MN-3 antibody is selected from the group consistingof amino acid residue 27, 30, 67, 68, 69 and 94 of the murine MN-3 heavychain variable region sequence (SEQ ID No. 11) or amino acid residue 20,22, 39, 60, 70 and 100 of the murine MN-3 light chain variable regionsequence (SEQ ID No. 9).
 5. The chimeric antibody or fragment thereof ofclaim 1, wherein the antibody or fragment thereof comprises the aminoacid sequences of cMN-3VK (SEQ ID NO:13) and cMN-3VH (SEQ ID NO:15). 6.The humanized antibody or fragment thereof of claim 2, wherein theantibody or fragment thereof comprises the amino acid sequences ofhMN-3VK (SEQ ID NO:18) and hMN-3VH (SEQ ID NO:21).
 7. The chimeric orhumanized antibody or fragment thereof claim 1, wherein the fragment isselected from the group consisting of Fv, F(ab′)₂, Fab′ and Fab.
 8. Thechimeric or humanized antibody or fragment thereof of claim 1, whereinthe antibody or fragment is bound to at least one diagnostic/detectionagent or at least one therapeutic agent or is part of a fusion protein.9. The chimeric or humanized antibody or fragment thereof of claim 8,wherein the diagnostic/detection agent comprises a photoactivediagnostic/detection agent, a chromagen or dye, a radionuclide with anenergy between 20 and 10,000 keV, a gamma-, beta- or a positron-emittingisotope, a contrast agent, a paramagnetic ion, an ultrasound-enhancingagent, a liposome or a radiopaque compound.
 10. The chimeric orhumanized antibody or fragment thereof of claim 8, wherein thetherapeutic agent is selected from the group consisting of aradionuclide, boron, gadolinium or uranium atoms, an immunomodulator, acytokine, a hormone, a hormone antagonist, an enzyme, an enzymeinhibitor, a photoactive therapeutic agent, a cytotoxic drug, a toxin,an angiogenesis inhibitor, a different antibody and a combinationthereof.
 11. The chimeric or humanized antibody or fragment thereof ofclaim 10, wherein the drug is selected from the group consisting ofantimitotic, alkylating, antimetabolite, angiogenesis-inhibiting,apoptotic, alkaloid, COX-2-inhibiting and antibiotic agents andcombinations thereof.
 12. The chimeric or humanized antibody or fragmentthereof of claim 10, wherein the drug is selected from the groupconsisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antibiotics, enzymes, epipodophyllotoxins, platinum coordinationcomplexes, vinca alkaloids, substituted ureas, methyl hydrazinederivatives, adrenocortical suppressants, hormone antagonists, enzymeinhibitors, endostatin, taxols and other taxanes, camptothecins,doxorubicin, and a combination thereof.
 13. The chimeric or humanizedantibody or fragment thereof of claim 10, wherein the toxin is selectedfrom the group consisting of ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.
 14. The chimeric or humanized antibody orfragment thereof of claim 10, wherein the immunomodulator is selectedfrom the group consisting of a cytokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interferon (IFN), a stem cell growth factor, erythropoietin,thrombopoietin, an antibody and a combination thereof.
 15. The chimericor humanized antibody or fragment thereof of claim 14, wherein thelymphotoxin is tumor necrosis factor (TNF), the hematopoietic factor isan interleukin (IL), the colony stimulating factor is granulocyte-colonystimulating factor (G-CSF) or granulocyte macrophage-colony stimulatingfactor (GM-CSF), the interferon is interferons-α, -βor -γ, and the stemcell growth factor is designated “S1 factor”.
 16. The chimeric orhumanized antibody or fragment thereof of claim 14, wherein the cytokineis selected from the group consisting of IL-1, IL-2, IL-3, IL-6, IL-10,IL-12, IL-18, IL-21, interferon-γ, TNT-α and a combination thereof. 17.The chimeric or humanized antibody or fragment thereof of claim 10,wherein the radionuclide is selected from the group consisting of P-32,P-33, Sc-47, Fe-59, Cu-64, Cu-67, Se-75, As-77, Sr-89, Y-90, Mo-99,Rh-105, Pd-109, Ag-111, I-125, I-131, Pr-142, Pr-143, Pm-149, Sm-153,Th-161, Ho-166, Er-169, Lu-177, Re-186, Re-188, Re-189, Ir-194, Au-198,Au-199, Pb-211, Pb-212, Bi-213, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m,Pt-109, In-111, Sb-119, Ho-161, Os-189m, Ir-192, Dy-152, At-211, Bi-212,Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Fm-255 andcombinations thereof.
 18. An antibody fusion protein comprising a firsthumanized or chimeric antibody or fragment according to claim 1,attached to a second antibody or fragment.
 19. The antibody fusionprotein of claim 18, wherein the second antibody or fragment is ahumanized or chimeric antibody or fragment.
 20. The antibody fusionprotein of claim 18, wherein the second antibody or fragment binds to anantigen other than NCA90.
 21. The antibody fusion protein of claim 19,further comprising a diagnostic/detection or therapeutic agentconjugated to the fusion protein.
 22. The antibody fusion protein ofclaim 20, wherein the second antibody or fragment binds to agranulocyte-associated antigen.
 23. A kit useful for treating oridentifying diseased tissues in a subject comprising: (A) a bi-specificantibody or antibody fragment having at least one arm that specificallybinds a targeted tissue and at least one other arm that specificallybinds a targetable conjugate, wherein the one arm that specificallybinds a targeted tissue is a humanized or chimeric antibody or fragmentthereof according to claim 1; (B) a first targetable conjugate whichcomprises a carrier portion which comprises or bears at least oneepitope recognizable by the at least one other arm of the bi-speciflcantibody or antibody fragment, and one or more conjugated therapeutic ordiagnostic agents; and (C) optionally, a clearing composition useful forclearing non-localized antibodies and antibody fragments; and (D)optionally, when the therapeutic agent conjugated to the firsttargetable conjugate is an enzyme, (i) a prodrug, when the enzyme iscapable of converting the prodrug to a drug at the target site; or (ii)a drug which is capable of being detoxified in the subject to form anintermediate of lower toxicity, when the enzyme is capable ofreconverting the detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of the drug at the target site, or(iii) a prodrug which is activated in the subject through naturalprocesses and is subject to detoxification by conversion to anintermediate of lower toxicity, when the enzyme is capable ofreconverting the detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of the drug at the target site, or(iv) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by the atleast one other arm of the bi-specific antibody or antibody fragment,and a prodrug, when the enzyme is capable of converting the prodrug to adrug at the target site.
 24. The kit of claim 23, wherein the targetableconjugate is selected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQ ID NO: 7); (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2; (iv)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2; (v)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vi)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (vii)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (viii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2; (ix)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)- NH2; (x)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xi)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2; (xii)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2; (xiv)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2; (xv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvi)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2; (xvii)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2; (xviii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2; (xix)Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(TscG-Cys)-NH2; (xx)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(TscG-Cys)-NH2;